And compositions for the analysis of nucleic acid molecules using size techniques

专利摘要:
Tack and linkers specifically designed for various nucleic acid reactions are described which are suitable for various nucleic acid reactions that require separation of nucleic acid molecules based on size.
公开号:KR19990081924A
申请号:KR1019980705637
申请日:1997-01-23
公开日:1999-11-15
发明作者:네스 제프리 반；존 씨 테이본；제이 제프리 하우버트；존 티 멀리건
申请人:니아리 린다 제이；라피진, 인코포레이티드；
IPC主号:

专利说明:

And compositions for the analysis of nucleic acid molecules using size techniques
Detection and analysis of nucleic acid molecules are among the most important technologies in biology. These techniques exist in the heart of molecular biology and play a role in rapidly expanding to the rest of the biology.
In general, one form of analysis of nucleic acid reactions involves the separation of nucleic acid molecules based on length. For example, the polymerase chain reaction (PCR) (see U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159), a widely used technique, identifies sequences present in a sample and provides a DNA molecule Has been a widely used technique for synthesizing the < RTI ID = 0.0 >
Briefly, in PCR, the DNA sequence is amplified by an enzymatic reaction that synthesizes a new DNA strand in a geometric or linear fashion. After amplification, the DNA sequence is detected and confirmed. Due to the need for unspecific amplification or purity that confound the assay, the PCR reaction products are generally separated prior to detection. Separation based on the size (i.e., length) of the product provides the most useful information. A method that provides the highest degradation performance of nucleic acid molecules is electrophoretic separation. In this method, each individual PCR reaction is applied to a suitable gel and a voltage is applied. The number of samples that can be processed is limited by the number of wells in the gel. For most gel systems, about 10 to 64 samples can be separated in a single gel. Thus, processing multiple samples is labor intensive and material intensive.
Electrical dynamic separation should be coupled to some detection system to obtain data. Nucleic acid detection systems use conventional and nearly exclusively intercalating dyes or radioactive labels, and rarely non-radioactive labels are used. The sample for use is an intercalating dye such as ethidium bromide. The dye is included in the gel matrix during electrophoresis, or after electrophoresis, the gel is adsorbed in a solution containing the dye. Dyes may be visible in some cases, but more frequently directly, and in particular emit fluorescence by light (e. G. UV) against tin bromide. Despite these apparently easy uses, each dye has some obvious disadvantages. First, dyes are not intense and large quantities of nucleic acid molecules must be present to show the product. Second, dyes typically induce mutagenesis or cancer.
Detection techniques that are more sensitive than dyes use radioactive (or non-radioactive) labels. Typically, radiolabeled nucleotides or radiolabeled primers are included in the PCR reaction. After separation, the radiation label is " visualized " by an autoradiography graph. Despite being more sensitive, detection has film limitations such as anti-deviation and nonlinearity. This limit can be overcome by detecting the label by image analysis of phosphorus. However, the radiolabel increases the source utilization and meets requirements that require specialized equipment and manpower training. For this reason, the use of non-radiative active labels has become popular. In such a system, the nucleotide can be labeled with a label such as a fluorescent moiety, biotin or digoxin that can be detected by an antibody or other molecule (e.g., a member of another ligand pair) labeled with an enzyme that reacts with a chromogenic substrate . Such a system does not present a safety problem as described above, but forms a high background (i. E., Low signal to noise ratio), using ingredients that can often change and cause unspecific reactions.
The present invention provides novel compositions and methods that can be used in a wide range of nucleic acid reactions and provides other related advantages.
As mentioned briefly, the present invention provides compositions and methods that can be used for a broad range of ligand pair reactions, which require size-based separation of molecules of interest, such as nucleic acid molecules. Representative examples of methods that may be improved in the context of the present disclosure include PCR, deviation display, RNA fingerprinting, PCR-SSCP, oligonucleotide assays, (E. G., A test based on exo- and endo-nuclease), and dideoxy fingering. The methods described herein can be utilized in a wide variety of fields including, for example, clinical diagnostic or investigational diagnostics, polymorphic determination and development of genetic maps.
In one aspect of the invention, methods comprise: (a) forming a nucleic acid molecule tacked from one or more selected target nucleic acid molecules, wherein the tack is interrelated to a particular nucleic acid fragment and detected by non-fluorescent spectroscopy or potentiometry Can be detected; (b) separating said tagged fragments by size; (c) cutting the tack from the tacked piece; (d) determining the identity of the nucleic acid molecule, including detecting the tag by non-fluorescence spectroscopy or potentiometry, and determining the identity of the nucleic acid molecule therefrom.
In a related aspect of the present invention, the methods comprise the steps of: (a) combining a nucleic acid probe tapped for a sufficient time and under conditions that allow hybridization of a nucleic acid probe tacked to a complementarily selected target nucleic acid sequence, Wherein the nucleic acid probe to which the tag is attached can be detected by non-fluorescence spectroscopy or potentiometry; (b) varying the size of the hybridized and tapped probe, the size of the unhybridized probe or target molecule, or the size of the probe: target hybrid; (c) separating the tacked probe by size; (d) cutting the tack from the tacked probe; (e) detecting a tack by a non-fluorescence spectrometry or a potentiometric method, and then detecting a nucleic acid molecule selected therefrom.
In a further aspect, the methods comprise: (a) forming a nucleic acid molecule tacked from a selected target molecule, wherein the tack correlates with a particular fragment and can be detected by non-fluorescent spectroscopy or potentiometric methods; (b) separating the set molecule by the length of the sequence; (c) cutting the tack from the molecule with the tack set; (d) detecting a tack by non-fluorescence spectroscopy or potentiometric methods, and then determining the genotype of the organism from which the gene form of the selected organism is determined.
In another embodiment, the methods comprise: (a) combining nucleic acid molecules with a selected target molecule for a sufficient time and under conditions that allow for hybridization of molecules set tacked to the target molecule, And can be detected by non-fluorescence spectroscopy or potentiometry; (b) separating the tagged fragment by the length of the sequence; (c) cutting the tack from the tacked piece; (d) detecting a tack by non-fluorescence spectroscopy or potentiometric methods, and then determining the genotype of the organism from which the gene form of the selected organism is determined.
In the context of the present invention, a "biological sample" is a sample that is obtained from living organisms (eg, mammals, fish, bacteria, parasites, viruses, fungi, etc.) or from the environment (E.g., phage library, organic molecular library, genomic clone, cDNA clone, and RNA clone, etc.) that can be produced by the method of the present invention, as well as the sample, artificially or synthetically. Representative examples of biological samples include biological milieu (e.g., blood, semen, cerebrospinal fluid, urine), biological cells (e.g., suprarenal cells, B or T cells, liver cells, fibroblasts, etc.) do. Finally, representative examples of organisms that can determine the genotype are virtually any single cell or multicellular organism, such as a warm-blooded animal, a mammal or a vertebrate animal (e.g., a human, a chimpanzee, a short tailed monkey, , Cells from cows, pigs, sheep, dogs, cats, rats, and mice as well as any of these animals), bacteria, parasites, viruses, fungi, and plants.
In various embodiments of the methods described above, the nucleic acid probes and / or molecules of the present invention may be formed, for example, by linkage, truncation or extension (e.g., PCR) reactions. In other related embodiments, the nucleic acid probe or molecule may be tacked with an oligonucleotide primer (such as a 5'-tacked oligonucleotide primer) or a dideoxynucleotide terminator set to a non-3 'tack .
In other embodiments of the present invention, the 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 250, 450, or more than 500 different, unique, tacked molecules can be used simultaneously in a given reaction where each tack can be identified individually and distinctly for a selected nucleic acid molecule or fragment, or probe.
In a further embodiment of the present invention, the tag (s) can be detected by fluorescence analysis, mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy or by constant potential current methods (e.g. using an electrical detector and a current detector) Can be detected. Representative examples of suitable spectrometric techniques include time-of-flight mass spectrometry, quadrupole mass spectrometry, magnetic sector mass spectrometry and electrical sector mass spectrometry. Specific embodiments of such techniques include ion-trap mass spectrometry, electrospray ionization mass spectrometry, ion spray mass spectrometry, liquid ionization mass spectrometry, atmospheric pressure ionization mass spectrometry, electron ionization mass spectrometry, Spectroscopic analysis, MALDI mass spectrometry, photoionization time-of-flight mass spectrometry, laser droplet mass spectrometry, MALDI-TOF mass spectrometry, APCI mass spectrometry, nano-spray mass spectrometry, Mass spectrometry, chemical ionization mass spectrometry, resonance ionization mass spectrometry, secondary ionization mass spectrometry and thermal spray mass spectrometry.
In other embodiments of the invention, the target molecule, hybrid and tacked probe, non-hybridized probe or target molecule, probe: target hybrid or tacked nucleic acid probe or molecule can be characterized by the size of the molecule One of the dimension dimensions), which is different from the method of using other molecules. Representative examples of such methods include gel electrophoresis, capillary electrophoresis, microchannel electrophoresis, HPLC, size exclusion chromatography, filtration, polyacrylamide gel electrophoresis, liquid chromatography, reverse phase exclusion chromatography, ion exchange chromatography , Reversed phase liquid chromatography, pulse electrophoresis, long-reverse electrophoresis, dialysis, and fluorescence-activated liquid dot classification. Also, a target molecule, a hybrid and tacked probe, a nonhybridized probe or target molecule, a probe: a target hybrid or a tacticized nucleic acid probe or molecule can be prepared using a solid support (e.g., hollow fibers (manufactured by Amicon Corporation, Danvers, Mass.), Beads (manufactured by Polysciences, Warrington, Pa.), Magnetic beads (manufactured by Robbin Scientific, Mountain View, Calif.), Plates, dishes and flasks (manufactured by Corning Glass Works, Corning, NY) (Becton Dickinson, Mountain View, Calif.), Screens and solid fibers (Edelman et al., US Pat. No. 3,843,324; Kuroda etal., US Pat. No. 4,416,777) , Mass.), And dip sticks. The first or second member, or the exposed nucleic acid, when bound to a solid support, further comprises washing the solid support of the unbound material in certain embodiments of the invention described herein.
In an embodiment, the tacified nucleic acid molecule or probe is chemically cleaved by methods such as an oxidation method, a reduction method, an acid instability method, a base instability method, an enzymatic method, an electrochemical method, a thermal method and a light instability method . In a further embodiment, the separation, cutting and detection steps can be performed in a continuous manner, for example, in a single apparatus that can be automated.
In certain embodiments of the present invention, the size of the hybrid and tacked probe, the non-hybridized probe or the target molecule or probe: the target hybrid is selected from polymerase extension, linking, exonuclease digestion, endonuclease digestion, restriction Is altered by a method selected from the group consisting of enzymatic degradation, site-specific recombinase degradation, ligation, mismatch-specific nuclease degradation, methylation-specific nuclease degradation, covalent attachment of probes to a target, and hybridization.
The methods or compositions described herein may be used for diagnostics, discussion, identification, developmental biology, biology, molecular medicine, toxicology, animal breeding, for example, PCR amplicon, RNA fingerprinting, Can be used in a wide variety of applications, including detection, dideoxy fingerprinting, restriction map and restriction fragment length polymorphism, DNA fingerprinting, gene form determination, mutation detection, oligonucleotide linkage analysis, sequence specific amplification have.
These and other aspects of the present invention will become more apparent from the following detailed description and the accompanying drawings. In addition, various references are set forth below in the description of a particular process or composition (e.g., a plasmid, etc.) and are incorporated by reference in their entirety.
The present invention relates generally to methods and compositions for analyzing nucleic acid molecules and more specifically to tags that can be used in a wide range of nucleic acid reactions requiring separation of nucleic acid molecules based on size.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a process flow diagram for the synthesis of a pentafluorophenyl ester of a mass spectrometric tack that can be chemically cleaved to separate tack using a carboxyl amide end.
Figure 2 is a work flow diagram for the synthesis of a pentafluorophenyl ester of a mass spectrometric tack that can be chemically cleaved to separate the tack using a carboxylic acid end.
Figures 3 to 6 and 8 are work flow diagrams for the synthesis of a set of tetrafluorophenyl esters of 36 mass spectrometry taps that can be photochemically cleaved.
Figure 7 is a process flow diagram for synthesis of a set of 36 amine-terminated photochemically cleavable mass spectrometry taches.
Figure 9 is a synthesis of 36 oligonucleotides with a photochemically cleavable mass spectrometry tag made from a corresponding set of 36 tetrafluorophenyl esters of photochemically cleavable mass spectrometry taxanes .
Figure 10 is a schematic representation of 36 oligonucleotides set with photochemically cleavable mass spectrometry taps prepared from a corresponding set of 36 amine-terminated photochemically cleavable mass spectrometry taches.
Figure 11 shows the simultaneous detection of multiple tags by mass spectrometry.
Figure 12 shows the mass spectrometry of only one alpha-cyano matrix.
13 shows a nucleic acid fragment to which a tag constructed by a module is set.
As indicated above, the present invention provides compositions and methods for analyzing nucleic acid molecules that require separation of nucleic acid molecules based on size. The present invention allows the simultaneous detection of molecules of interest, including nucleic acids and fragments, proteins, peptides, and the like.
In one briefly mentioned embodiment, the invention provides a compound wherein the molecule of interest or its precursor is bound to the tack by unstable (or unstable) bonds. Accordingly, the compounds of the present invention can be represented by the following formula (1)
T-L-X
In this formula,
T is a tack element,
L is a linker containing an unstable or unstable linkage,
X is the molecule of interest (MOI) component or functional group component (L _h ) through which the MOI can be coupled to the TL.
Accordingly, the compounds of the present invention can be represented more specifically by the following formulas (2) and (3).
T-L-MOI
TLL _h
For reasons that will be described in detail below, the set of T-L-MOI compounds can be separated from the residue of the compound by subjecting it to conditions which deliberately cause the destabilizing bond (s) to be cleaved. The tack residue is then characterized by one or more analytical techniques that provide direct information about the structure of the tack residue and (most importantly) indirect information about the identity of the corresponding MOI.
As a sample of a representative compound of the present invention wherein L is a direct bond, reference is made to the following structure (i):

In structure (i), T is a polycyclic aromatic residue containing a nitrogen bonded to a carbonyl group, X is an MOI (and specifically a nucleic acid fragment terminating in an amine group), L is an amide group It is a combination. Since the amide bond is unstable to the bond in T, as revealed in the prior art, the amide bond can be chemically decomposed (destroyed) by acidic or basic conditions which leave the bond in the tacky component unchanged. Thus, the tack residue (i.e., the cleavage product containing T) can be released as shown below:

However, the bond (L) may be more than a simple direct bond as shown in the following examples, with reference to other representative compounds of the present invention having the structure (ii) shown below.

Compounds having ortho-nitrobenzylamine residues (see squared atoms in structure (ii)) are photodegradable and unstable, so that exposure of these compounds to radiation of a particular wavelength results in the formation of benzylamine bonds (see structure Lt; RTI ID = 0.0 > a < / RTI > bond). Thus, structure (ii) has the same T and MOI groups as in structure (i), but the linker group contains multiple elements and bonds, especially where unstable bonds are present. Thus, a tack residue (residue containing T) is released from the residue of the compound by photolysis of the structure (ii) as shown below.

Thus, the present invention provides compounds that upon cleavage under suitable cleavage conditions, cleavage reactions occur to release the tack residue from the residue of the compound. The compounds of the invention may be described in terms of tack residue, MOI (or precursor thereof, L _h ), and the unstable linkage (s) to which the two groups are attached together. In addition, the compounds of the present invention can be described in terms of ingredients from which they are formed. That is, the compounds of the present invention can be described as reaction products of the following tact reactants, linker reactants and MOI reactants.
The tack reactant comprises a chemical handle (T _h ) and various components (T _vc ), and the reactive reagent has the structure of Formula (4)
T _vc -T _h
To illustrate this nomenclature, reference can be made to structure (iii) which represents a tact reactant which can be used to prepare compounds of structure (ii). The reactive reactants having structure (iii) may contain tack modified components and tack handles as shown below:

In structure (iii), the tackle handle (-C (= O) -A) simply provides a path for reacting the tack reactant with the linker reagent to form the T-L residue. In structure (iii), group " A " indicates that the carboxyl group is in a chemically active state and is ready for coupling with other handles. &Quot; A " may be, for example, hydroxy group or pentafluorophenoxy among many other possibilities. The present invention provides a number of possible tackle handles that can be combined with tack strain components as discussed in detail below. The tack modification component is a moiety " T " in the formula T-L-X and may also be part of the tack residue formed from the reaction of cleaving L.
As will be discussed in detail below, the tackifier component is intended for use in the preparation of a set of compounds according to the present invention in which the members of the set possess unique strain components, so that individual members can be distinguished from each other by analytical techniques . In one embodiment, the tack strain component of structure (iii) can be a member of one of the following sets, wherein the members of the set can be distinguished by its UV or mass spectrum:

Similarly, linker reactants may be described in terms of chemical handles (essentially two or more are present, each of which may be represented by L _h ) on the side of the binding instability component, wherein the linker instability component has the requisite instability (L ² ) and any unstable moieties (L ¹ and L ³ ), and any unstable moiety contributes efficiently to separate L ² from the handle L _h , and the essential unstable moiety is in the linker-unstable moiety Contributes to providing instability bonds. Thus, the linker reagent can be represented by the following formula 5:
L _h -L ¹ -L ² -L ³ -L _h
The nomenclature used to describe the linker reagent can be shown in terms of structure (iv) again from the compound of structure (ii):

As shown in structure (iv), an atom can contribute to one or more functional roles. Thus, in structure (iv), the benzyl nitrogen acts as a chemical handle to allow the linker reactant to bind to the reactive reagent by the amide formation reaction, and the benzylic carbon- Contributes as a necessary part of the structure of the labile residue L < ^{2 &} gt ;. Structure (iv) also indicates that the linker reactant can retain the L ³ group, in this case the methylene group, even though the L ¹ group does not. Similarly, the linker reactant may have the L ¹ group but not the L ³ group, or may have the L ¹ group and the L ³ group, or may not have the L ¹ group or the L ³ group. In structure (iv), the presence of group " P " next to the carbonyl group indicates that the carbonyl group is protected from the reaction. When such an arrangement is provided, the activated carboxyl group of the taxane reactant (iii) can react with the amine group of the linker reactant (iv) to form an amide bond and provide a compound of the formula TLL _h .
The MOI reactant is an appropriate reactive form of the molecule of interest. When the molecule of interest is a nucleic acid fragment, a suitable MOI reactant is a nucleic acid fragment bound to its alkylene chain terminating in the amino group followed by a phosphodiester group via its 5 ' hydroxy group. This amino group is reacted with a carbonyl group of structure (iv) (of course after deprotecting the carbonyl group, preferably after successive activation of the carbonyl group towards the reaction with the amine group) .
In view of the temporal order, the present invention provides a process for the preparation of a compound of formula (I), which comprises reacting a tack reactant (having a chemical tackle handle and a tacky labile component), a linker reagent (having two chemical linker handles and an essential labile moiety and 0 to 2 optional labile moieties) MOI reactants (with molecules of the component of interest and chemical molecules of the handle of interest) to form the TL-MOI. Thus, in order to form a TL-MOI, selecting reactants and by first reaction with a linker reagent provides a TLL _h, which was the MOI reactant reaction and TLL _h providing a TL-MOI, or (but not substantially preferably) the linker reactant and the to a first reaction with a MOI reactant provide L _h -L-MOI, and then provides a response and select the L _h -L-MOI reactant to TL-MOI. For convenience, compounds having the formula TL-MOI can be described in terms of tact reactants, linker reactants and MOI reactants that can be used to form such compounds. Of course, the same compounds of the formula TL-MOI can be prepared by other methods (typically more difficult) and still fall within the scope of the TL-MOI compounds of the present invention.
In any event, the present invention provides that the TL-MOI compound is placed under cleavage conditions so that the tack residue is released from the residue of the compound. The tack residue can comprise at least a tacky instability component and is typically present in some or all of the atom from the tackle handle, some or all atoms from the linker handle used to bind the tack reactant to the linker reactant, TL-MOI , It may further comprise an optional labile residue L < ¹ > and may contain a portion of the required labile residue L < ² > depending on the exact structure of L < ² & Conveniently, tack residues can be referred to as residues containing T, since T typically constitutes the main fragment (in terms of mass) of the tack residue.
In providing such an introduction to one aspect of the present invention, the various components T, L, and X can be described in detail. These techniques then begin with the definition of certain terms that can be used to describe T, L, and X.
As used herein, the term " nucleic acid fragment " refers to a molecule that replenishes (i.e., replenishes all or part of) a selected target nucleic acid molecule and includes a naturally or non- , By synthesis or by recombination, and where appropriate may be in the form of a double strand or a single strand; Oligonucleotides extending in the 5 'to 3' direction by an oligonucleotide (e.g., DNA or RNA), a primer, a probe, a nucleic acid analog (e.g., PNA), a polymerase, A nucleic acid, a nucleic acid capped at the 3 ' or 5 ' end with a compound terminated by a dideoxy terminator or preventing polymerization at the 5 ' or 3 ' end, and combinations thereof. Supplementation of a nucleic acid fragment to a selected target nucleic acid molecule generally means the appearance of at least about 70% specific base pairs through the length of the fragment. Preferably, the nucleic acid fragment comprises at least about 80% of a specific base pair; Most preferably at least about 90%. The assays for measuring the percentage of mismatch (and a specific sorghum pair of percentages) are well known in the art and are based on percent mismatches as a function of Tm when referenced to a complete base pair control.
As used herein, the term " alkyl ", alone or in combination, refers to a saturated, straight or branched hydrocarbon radical containing from 1 to 10, preferably from 1 to 6 and more preferably from 1 to 4 carbon atoms will be. Examples of such radicals include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert- butyl, pentyl, iso-amyl, . The term " alkylene " refers to a saturated, straight or branched chain hydrocarbon radical containing from 1 to 10, preferably from 1 to 6, more preferably from 1 to 4 carbon atoms. Examples of such radicals include, but are not limited to, methylene, ethylene (-CH ₂ -CH ₂ -), propylene, and the like.
The term " alkenyl ", alone or in combination, refers to straight or branched chain hydrocarbon radicals having at least one carbon-carbon double bond in the range of from 2 to 10, preferably from 2 to 6, more preferably from 2 to 4 carbon atoms . Examples of such radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z- isobutenyl, E- and Z- And the like. The term " alkenylene " refers to straight or branched hydrocarbon di-radicals having from 2 to 10 carbon atoms, preferably from 2 to 6 carbon atoms, more preferably from 2 to 4 carbon atoms, having at least one carbon- carbon double bond. Examples of such radicals include, but di doejin limited to this, and the like methylidene (= CH _2), ethylidene (-CH = CH-), propylidene (-CH ₂ -CH = CH-).
The term " alkynyl ", alone or in combination, refers to straight or branched chain hydrocarbon radicals having from 2 to 10, preferably from 2 to 6 and more preferably from 2 to 4 carbon atoms having at least one carbon-carbon triple bond . Examples of such radicals include, but are not limited to, ethynyl (acetylenyl), propynyl (propargyl), butynyl, hexynyl, decynyl, and the like. The term " alkynylene ", alone or in combination, refers to straight or branched chain hydrocarbon radicals having from 2 to 10 carbon atoms, preferably from 2 to 6 carbon atoms, more preferably from 2 to 4 carbon atoms, having at least one carbon- . Examples of such radicals include, but are not limited to, ethynylene (-C C-), propynylene (-CH ₂ -C C-), and the like.
The term " cycloalkyl ", alone or in combination, refers to a saturated cyclic arrangement of carbon atoms of 3 to 8 membered rings, preferably 3 to 6 membered rings. Examples of such cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term " cycloalkylene " refers to the di-radical form of a cycloalkyl.
The term " cycloalkenyl ", alone or in combination, refers to a cyclic carbocycle containing from 4 to 8, preferably 5 or 6, carbon atoms and having one or more double bonds. Examples of such cycloalkenyl radicals include, but are not limited to, cyclopentenyl, cyclohexenyl, cyclopentadienyl, and the like. The term " cycloalkenylene " refers to the di-radical form of a cycloalkenyl.
The term " aryl " refers to carbocyclic (consisting entirely of carbon and hydrogen) aromatic groups selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl and anthracenyl; Or a group selected from the group consisting of furyl, thienyl, pyridyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, Thiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, Benzo [b] thiophenyl, 1H-indazolyl, benzo [b] furanyl, benzo [b] thiophenyl, Benzimidazolyl, benzthiazolyl, furanyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1,8- Refers to a heterocyclic aromatic group selected from the group consisting of aryl, heteroaryl, heteroaryl, heteroaryl, heteroaryl, heteroaryl, heteroaryl,
As defined herein, is independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, cyano, carboxy, carboalkoxy, Alkoxy, alkenoxy or alkynoxy, alkylamino, alkenylamino, alkynylamino, aliphatic or aromatic acyl, alkoxy-carbonylamino, alkylsulfonylamino, morpholinocarbonylamino, Thiomorpholinocarbonylamino, N-alkyl guanidino, aralkylaminosulfonyl, and the like; Aralkoxyalkyl; N-aralkoxyurea; N-hydroxylurea; N-alkenyl urea; N, N- (alkyl, hydroxyl) urea; Heterocyclyl; Thioaryloxy-substituted aryl; N, N- (aryl, alkyl) hydrazino; Ar'-substituted sulfonylheterocyclyl; Aralkyl-substituted heterocyclyl; Cycloalkyl and cycloalkenyl-substituted heterocyclyl; Cycloalkyl-fused aryl; Aryloxy-substituted alkyl; Heterocyclylamino; Aliphatic or aromatic acylaminocarbonyl; Aliphatic or aromatic acyl-substituted alkenyl; Ar'-substituted aminocarbonyloxy; Ar ', Ar'-disubstituted aryl; Aliphatic or aromatic acyl-substituted acyl; Cycloalkylcarbonylalkyl; Cycloalkyl-substituted amino; Aryloxycarbonylalkyl; Lt; RTI ID = 0.0 > phosphodiamidic < / RTI > acid or ester.
The term " Ar " " refers to hydrogen, halogen, hydroxy, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, 1,2-dioxymethylene, Alkoxy, alkenoxy, alkoxy, alkenoxy, alkynoxy, alkylamino, alkenylamino or alkynylamino, alkylcarbonyloxy, aliphatic or aromatic acyl, alkylcarbonylamino, alkoxycarbonylamino, alkylsulfonylamino, N, N-dialkylurea, and N, N-dialkylurea.
The term " alkoxy ", alone or in combination, refers to an alkyl ether radical as defined above for the term " alkyl ". Examples of suitable alkyl ether radicals include but are not limited to methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, .
The term " alkenoxy ", alone or in combination, refers to a radical of alkenyl-O- as defined above, provided that the term " alkenyl " Examples of suitable alkenoxy radicals include, but are not limited to, allyloxy, E- and Z-3-methyl-2-propenoxy, and the like.
The term " alkynyloxy ", alone or in combination, refers to a radical of an alkynyl-O- as defined above, provided that the term " alkynyl " Examples of suitable alkynoxy radicals include, but are not limited to, propargyloxy, 2-butynyloxy, and the like.
The term " thioalkoxy " refers to a thioether radical of alkyl-S- wherein alkyl is as defined above.
The term " alkylamino ", alone or in combination, refers to a mono- or di-alkyl-substituted amino radical as defined above (i.e. a radical of alkyl-NH- or (alkyl) ₂ -N-) I will mention. Examples of suitable alkylamino radicals include, but are not limited to, methylamino, ethylamino, propylamino, isopropylamino, t-butylamino, N, N-diethylamino and the like.
The term " alkenylamino ", alone or in combination, refers to an alkenyl-NH- or (alkenyl) ₂ N- radical as defined above, provided that the radical " alkenyl " . An example of such an alkenylamino radical is an allyl amino radical.
The term " alkynylamino ", alone or in combination, refers to an alkynyl-NH- or (alkynyl) ₂ N- radical as defined above, provided that the radical " alkynyl " An example of such an alkynylamino radical is a propargylamino radical.
The term " amide " refers to the group -N (R ¹ ) -C (═O) - or -C (═O) -N (R ¹ ) - wherein R ¹ is defined herein, ). The term "substituted amide" refers to the state where R ¹ is not hydrogen, while the term "unsubstituted amide" refers to the state where R ¹ is hydrogen.
The term " aryloxy ", alone or in combination, refers to an aryl-O- radical as defined above. Examples of aryloxy radicals include, but are not limited to, phenoxy, naphthoxy, pyridyloxy, and the like.
The term " arylamino ", alone or in combination, refers to an aryl-NH- radical as defined above. Examples of arylamino radicals include, but are not limited to, phenylamino (anilido), naphthylamino, 2-, 3-, and 4-pyridylamino, and the like.
The term " aryl-fused cycloalkyl ", alone or in combination, refers to a cycloalkyl radical in which the terms " cycloalkyl " and " aryl " define two adjacent elements with an aryl radical, as defined above. An example of an aryl-fused cycloalkyl radical is a benzo-fused cyclobutyl radical.
The term " alkylcarbonylamino ", alone or in combination, refers to a radical of alkyl-CONH, wherein the term " alkyl "
The term " alkoxycarbonylamino ", alone or in combination, refers to a radical of alkyl-OCONH, as defined above for the term " alkyl ".
Alone or in combination, the term "alkylsulfonyl amino" to the term "alkyl" refers to a radical of the same, -SO ₂ NH-alkyl as defined above.
The term " arylsulfonylamino ", alone or in combination, refers to a radical of aryl-SO ₂ NH, as defined above.
The term " N-alkylurea ", alone or in combination, refers to a radical of alkyl-NH-CO-NH as defined above for the term " alkyl "
The term " N-arylurea ", alone or in combination, refers to a radical of alkyl-NH-CO-NH as defined above for the term " aryl ".
The term " halogen " means fluorine, chlorine, bromine and iodine.
The term " hydrocarbon radical " refers to an arrangement of carbon and hydrogen atoms in which only a single hydrogen atom is required to be an independently stable molecule. Thus, a hydrocarbon radical has one open valence site on the carbon atom through which the hydrocarbon radical can be attached to the other atom (s). Examples of hydrocarbon radicals are alkyl, alkenyl, cycloalkyl, and the like.
The term " hydrocarbon di-radical " refers to the arrangement of carbon and hydrogen atoms in which two hydrogen atoms are required to be independently stable molecules. Thus, a hydrocarbon radical has two open valence sites on one or two carbon atoms through which a hydrocarbon radical can be attached to another atom (s). Examples of hydrocarbon di-radicals are alkylene, alkenylene, alkynylene, cycloalkylene, and the like.
The term " hydrocarbyl " refers to any stable arrangement consisting solely of carbon atoms and hydrogen atoms having a single atomic valence moiety coupled to another moiety, so that alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl An arylalkyl, an alkylaryl, and the like. Hydrocarbon radicals are another name for hydrocarbons.
The term " hydrocarbylene " refers to any stable arrangement consisting solely of carbon atoms and hydrogen atoms having two valence moieties bonded to other moieties, so that alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene , Arylene (in which no heteroatom is incorporated in the arylene ring), arylalkylene, alkylarylene, and the like. Hydrocarbon di-radicals are another name for hydrocarbylene.
The term " hydrocarbyl-O-hydrocarbylene " refers to a hydrocarbyl group bonded to an oxygen atom, wherein an oxygen atom is replaced by a hydrocarbyl group at one of the two valence points at which the hydrocarbylene group is bonded to the other moiety Lt; RTI ID = 0.0 > group. The terms "hydrocarbyl-S-hydrocarbylene", "hydrocarbyl-NH-hydrocarbylene" and "hydrocarbyl-amide-hydrocarbylene" have the same meaning, Group.
The term N- (hydrocarbyl) hydrocarbylene refers to a hydrocarbylene group in which one of the two valence moieties is bonded to a nitrogen atom and the nitrogen atom is simultaneously bonded to a hydrogen and a hydrocarbyl group. The term N, N-di (hydrocarbyl) hydrocarbylene refers to a hydrocarbylene group in which one of the two valence moieties is bonded to a nitrogen atom and the nitrogen atom simultaneously bonds to two hydrogens and two hydrocarbyl groups will be.
The term " hydrocarbyl acyl-hydrocarbylene " refers to a hydrocarbyl group that is attached via an acyl (-C (= O) -) group to one of the two valence moieties of the hydrocarbylene group.
The terms " heterocyclyl hydrocarbyl " and " heterosilyl " refer to a stable cyclic arrangement of carbon atoms and atoms comprising up to four atoms (referred to as heteroatoms) selected from oxygen, nitrogen, I will mention. The cyclic arrangement may be in the form of a 3 to 7 membered monocyclic ring or an 8 to 11 membered non-cyclic ring. The ring can be saturated or unsaturated (including aromatic rings) and optionally benzo-fused. In the ring, nitrogen and sulfur may be present in any oxidation form, including the quaternized form of nitrogen. Heterocyclyl hydrocarbyl may be attached to any endocyclic carbon or heteroatom that forms a stable structure. A preferred heterocyclyl hydrocarbyl includes a 5-7 membered monocyclic heterocycle containing one or two nitrogen heteroatoms.
Substituted heterocyclyl hydrocarbyl refers to a heterocyclyl hydrocarbyl as defined above wherein at least one of the reducing moieties is bonded to a pointed substituent that does not extend the ring.
In the context of hydrocarbyl and hydrocarbyl groups, the term " any of the aforementioned derivatives wherein one or more hydrogens is replaced by the same number of fluorine " refers to a molecule containing carbon, hydrogen and fluorine atoms but not other atoms I will mention.
The term " activated ester " is an ester containing a nucleophile such as an amine and a " leaving group " that can be quickly displaced by an alcohol nucleophile or a thiol nucleophile. Such leaving groups are well known and include, but are not limited to, N-hydroxysuccinimide, N-hydroxybenzotriazole, halogen (halides) including tetrafluorophenolate and thioalkoxy, alkoxy and the like do. The term " protected ester " refers to an ester group that is shielded or otherwise unreactive (see Greene, " Protecting Groups In Organic Synthesis ").
In the context of the foregoing definitions, other chemical terms used throughout this disclosure will be readily apparent to those skilled in the art. The terms may be used alone or in any combination thereof. Preferred and more preferred radical chain lengths apply to all such combinations.
A. Formation of a nucleic acid fragment
As noted above, one aspect of the invention provides a general approach for sequencing DNA that can use more than 16 taps in each lane; It is possible to detect the tack by continuous detection and to read the sequence according to size separation as in conventional fluorescent-based sequence analysis. This approach can be applied to any DNA sequencing technique based on the separation of the size of the molecule to which the tag is set. Methods for sequencing nucleic acids as well as suitable tack and linkers for use in the present invention are discussed in further detail below.
1. Choose
As used herein, " tag " refers generally to a chemical moiety that is used to uniquely identify a " molecule of interest ", and more specifically, to a tag moiety Lt; RTI ID = 0.0 > closest < / RTI >
The tack useful in the present invention possesses several functions, such as:
1) It can be distinguished from all other tack. This differentiation from other chemical moieties may be based on the chromatographic behavior of the tack, especially after the cleavage reaction, spectroscopic or potentiometric properties, or combinations thereof. Spectroscopic analytical methods that are usefully distinguished include mass spectrometry (MS), infrared (IR), ultraviolet (UV), and fluorescence (where MS, IR and UV are preferred, and MS is most preferred) .
As the potential difference method, an ammeter is most preferable.
2) tack can be detected when present in 10 ^-22 to 10 ^-6 moles
3) The tack holds a chemical handle that can be attached to an MOI that tends to be uniquely identified. May be attached directly to the MOI, or may be indirectly attached through a " linker " group.
4) The tack is chemically stable for all applied operations including cutting from attachment and MOI and manipulation of any MOI to which the tack is attached.
5) The tack does not significantly interfere with the operations performed at the MOI, while the tack attaches to it. For example, when a tack is attached to an oligonucleotide, the tack should not significantly interfere with any hybridization or enzymatic reaction (e.g., PDR sequencing) that takes place in the oligonucleotide. Similarly, when a tack is attached to an antibody, it should not significantly interfere with antigen recognition by the antibody.
The tack residue desired to be detected by a specific spectroscopic analysis or potential difference method possesses properties that improve the detection sensitivity and specificity by such a method. Typically, the tack residue will retain this property because it is typically designed as a tacking component that constitutes a major part of the tack residue. In the discussion that follows, the use of the word " tack " will typically refer to a tack residue (i.e., a cleavage product containing a tack variant component), but also typically includes tack residues responsible for providing uniquely detectable properties It may be considered to refer to the tacky strain component itself. In the compounds of formula TLX, the " T " moiety may contain tackifying moieties. If the tack strain component is designed to be characterized by, for example, mass spectrometry, the " T " portion of the TLX can be referred to as T ^ms . Similarly, a cleavage product from a TLX containing T may be referred to as a residue containing T ^ms . The following spectroscopic methods and potential difference methods can be used to characterize residues containing T ^ms .
a. Features of MS Tack
If the tack can be analyzed by mass spectrometry (i.e., MS-readable tack and also referred to herein as a residue containing MS tack or T ^ms ), an essential feature of the tack is that it can ionize it . Thus, this is a desirable element in the design of MS readable tack to incorporate chemical functional groups capable of holding a positive or negative charge under ionization conditions in MS. This feature provides an overall greater sensitivity of detection in ionization of improved efficiency and especially in electrospray ionization. The chemical functional group that supports the ionized charge can be derived from both T ^ms or L. Factors that can increase the relative sensitivity of analytes detected by mass spectrometry are described in Sunner, J., et al., Anal. Chem. 60: 1300-1307 (1988).
Preferred functional groups to facilitate retaining negative charge are organic acids such as phenolic hydroxyl, carboxylic acids, phosphonates, phosphates, tetrazoles, sulfonylureas, perfluoroalcohols and sulfuric acid.
Preferred functional groups for facilitating holding positive charges under ionizing conditions are aliphatic or aromatic amines. Examples of amine functional groups that provide improved detection performance of MS tack include quaternary amines (i.e., amines having four bonds to each carbon atom, see U.S. Pat. No. 5,240,859 to Aebersold) and tertiary Amines (i.e., amines having four bonds to each carbon atom, including the C = NC group as present in pyridine, see Anal. Biochem. 224: 373, 1995; Bures et al., Anal. Biochem 224: 364, 1995). Hindered tertiary amines are particularly preferred. The tertiary and quaternary amines may be alkyl or aryl. The residue containing T ^ms should have one or more ionizable species, but may have one or more ionizable species. The preferred charge state is one ionized species at a charge. Thus, it is preferred that the residues containing each T ^ms (each tacky component) contain only one hindered amine or organic acid group.
Suitable amine containing radicals capable of forming part of the residue containing T ^ms include:


Identification of the tack by means of mass spectrometry is preferably based on its molecular weight to charge ratio (m / z). The preferred molecular weight range for MS tack is about 100 to 2,000 dalton, preferably the mass of the residue containing T ^ms is at least about 250 dalton, more preferably at least about 300 dalton, and most preferably at least about 350 dalton. In general, mass spectrometry is difficult to distinguish among residues having ions present at less than about 200 to 250 dalton (depending on the precise mechanism), and thus the mass of residues containing the preferred T ^ms of the invention exceeds this range .
As described above, residues containing T ^ms may contain atoms other than atoms present in the tack strain component and are actually present in the T ^ms itself. Thus, the mass of T ^ms itself may be less than about 250 dalton, as long as the mass of the residue containing T ^ms is greater than about 250 dalton. Thus, the mass of T ^{ms may be in} the range of 15 (i.e., the methyl radical) to 10,000 dalton, preferably in the range of 100 to about 5,000 dalton, and more preferably in the range of about 200 to about 1,000 dalton.
It is relatively difficult to distinguish tack by mass spectrometry when incorporating atoms with one or more isotopes in significant amounts. Thus, a preferred T group intended for identity verification by mass spectrometry (T ^ms group) contains carbon, at least one hydrogen and fluorine, and any element selected from oxygen, nitrogen, sulfur, phosphorus and iodine. While other elements may be present at T ^ms , their presence may make the analysis of mass spectrometry data a little more difficult. Preferably the T ^ms group has only carbon, nitrogen and oxygen atoms besides hydrogen and / or fluorine.
Fluorine is any desired element in the T ^ms group. Fluoride is, of course, much heavier than hydrogen. Thus, the presence of fluorine atoms rather than hydrogen atoms results in a larger mass of the T ^ms group, thereby allowing the preferred T ^ms group to reach and exceed masses above 250 dalton, as described above. It also provides greater volatility in residues containing T ^ms by replacing hydrogen with fluorine, and the greater volatility of analytes is improved when mass spectrometry is used as a detection method.
The molecular formula T ^ms is C _1-500 N _0-100 O _0-100 S _0-10 P _0-10 H _α F _β I _δ where the sum of α, β and δ is the sum of the unsatisfied valences C, N, O, S, and P atoms). Name C _1-500 N _0-100 O _0-100 S _0-10 P _0-10 H _α F _β I _δ means that T ^ms contains one or more carbon atoms and has 100 nitrogen atoms, Means that it may contain any number of 1 to 500 carbon atoms besides optionally containing sulfur atoms and 10 phosphorus atoms ("N _{0 -} " means that T ^ms does not need to contain any nitrogen atoms ). The symbols α, β and δ represent the number of hydrogen, fluorine and iodine in T ^ms , where any two of these numbers may be zero, and the sum of these numbers is C, N, O, S and P atoms Lt; / RTI > is equal to the unsatisfied total valence of. Preferably, T ^ms is a C _1-50 N _0-10 O _0-10 H _α F _β , wherein the sum of α and β corresponds to the number of hydrogen and fluorine atoms respectively present in the residue. Respectively.
b. Features of IR tack
There are two main types of IR detection of organic chemistry groups: Raman scattering IR (IR) and adsorption IR. Raman scattering IR spectra and adsorption IR spectra are complementary spectroscopic analytical methods. In general, the Raman excitation depends on the polarization change of the bond, whereas the IR absorption depends on the change in dipole moment of the bond. A weak IR adsorption line becomes a strong Raman line and vice versa. The wavelength value is a characteristic unit of the IR spectrum. There are 3 spectral regions for IR tack having a source of IR from the IR, 600 to 30cm ^-1 at the individual application of, 12500 through 4000cm near-IR at the ^-1, 4000 to 600cm ^-1. A medium spectral region may be preferred for the applications mentioned herein if the compound can contribute to the MOI, probe or primer as a tag for identification purposes. For example, carbonyl groups (1850-1750 cm ^-1 ) can be measured for carboxylic acids, carboxylic acid esters and amides, and alkyl and aryl carbonate, carbamates and ketones. NH bending (1750 to 160 cm ^-1 ) can be used to identify the identity of amines, ammonium ions, and amides. At 1400 to 1250 cm <" ^{1 &} gt ;, CN extensions in amides as well as R-OH bending are detected. An aromatic substitution pattern is detected at 900-690 cm ^-1 (CH bending, NH bending to ArNH ₂ ). A nitrogen compound such as saturated CH, an olefin, an aromatic ring, a double and triple bond, an ester, an acetal, a ketal, an ammonium salt, an oxime, a nitro, an N-oxide and a nitrate, an azo, a hydrazone, a quinone, And lactams all possess vibrational infrared correlation data (Pretsch et al., Spectral Data for Structure Determination of Organic Compounds, Springer-Verlag, New York, 1989). Preferred compounds may include aromatic nitriles exhibiting very strong nitrile extended vibrations at 2230-2210 cm < ^-1 >. Other useful forms of the compounds are aromatic alkynes with strong elongation vibrations that cause a sharp adsorption band between 2140 and 2100 cm <" ^{1 &} gt ;. The third form of the compound is an aromatic azide exhibiting an intensive adsorption band in the 2160 to 2120 cm ^-1 region. Thiocyanate is a representative compound strongly adsorbed at 2275 to 2263 cm ^-1 .
c. Features of UV tack
Organic chromophore forms and their respective UV-visible properties are provided in Scott, Interpretation of the UV Spectra of Natural Products, Permagon Press, New York, 1962. A chromophore is an atom or group of atoms or electrons for adsorbing particular light. Experimental rules exist for π to π ^* maxima in the conjugate system (Pretsch et al., Spectral Data for Structure Determination of Organic Compounds, p. B65 and B70, Springer-Verlag, New York, 1989]. Preferred compounds (with a conjugated system) have n to ^* And To ^* Transitions. Examples of such compounds are acid violet 7, acridine orange, acridine yellow G, brilliant blue G, Congo red, crystal violet, malachite green oxalate, methanyl yellow, methylene blue, methyl orange, methyl violet B, Naphthol Green B, Oil Blue N, Oil Red O, 4-Phenyl Azophenol, Safran O, Solvent Green 3, and Sudan Orange G, both of which are commercially available (Aldrich, Milwaukee, WI). Other suitable compounds are described, for example, in Jane, I., et al., J, Chrom. 323: 191-225 (1985).
d. Characteristics of fluorescent tack
Fluorescent probes are most directly identified or measured by their hygroscopicity and fluorescence emission wavelength and intensity. Emission spectra (fluorescence and phosphorescence) are much more sensitive and allow more specific measurements than adsorption spectra. Other photophysical properties such as lifetime in the excited state and fluorescence anisotropy are used less widely. The most commonly useful intensity parameters are the molar filtration coefficient (ε) for adsorption and the quantum yield (QY) for fluorescence. The value of epsilon is specified at a single wavelength (typically the maximum absorption of the probe), whereas QY is a measure of the total photon emission over the entire fluorescence spectral profile. The width of the narrow optical bend (less than 20 nm) is typically used for fluorescence excitation (through adsorption), while the fluorescence detection bend width is increased from the entire spectrum for maximum sensitivity to narrow bends It can be varied. The fluorescence intensity for the probe molecule is proportional to the product of And QY. Among the currently significant fluorophore groups, the range of these parameters is about 10,000 to 100,000 cm ^-1 M ^-1 for ε and 0.1 to 1.0 for QY. Compounds that can be used as fluorescent tags are: fluorescein, rhodamine, lambda blue 470, lambda green, lambda red 664, lambda red 665, acridine orange, and propidium iodide, which are commercially available (Manufacturer: Lambda Fouorscence Co., Pleasant Gap, Pa.). Fluorescent compounds such as nile red, Texas Red, Li ssamin (lissamine ^TM) and Bodipy (BODIPY ^TM) are commercially available [roducer: Molecular Proves (Eugene, OR) ].
e. Characteristics of Potential Difference
The principle of electrochemical detection (ECD) is based on the oxidation or reduction of a compound that forms an electric current that can be measured and received by an electron at a particular applied voltage. When a specific compound is applied to the deviation of dislocation, the molecule is subjected to rearrangement of molecules while reducing (oxidizing) electrons at the working electrode surface or obtaining (reducing) electrons, and these compounds perform an electrical reaction and an electrochemical reaction . The EC detector applies a voltage to the surface of the electrode from which the HPLC eluant flows. The electroactive compound eluting from the column accepts (reduces) electrons to provide electrons or oxidize and form current peaks in real time. The amount of current formed depends on both the concentration of the analyte and the applied voltage using each compound having a specific voltage at which oxidation or reduction begins. Currently the most popular electrochemical detectors are amperometric detectors that measure the current produced from an electrochemical reaction while maintaining a constant potential difference. This type of spectroscopic analysis is now referred to as the " constant potential current method ". The ammeter is commercially available (manufacturer: ESA, Inc., Chelmford, Mass.).
If the detection efficiency is 100%, the characterized detector is named " electrical detector ". Of the detectors useful for assays, the electrical detectors with many practical advantages with respect to selectivity and sensitivity to this form are sensitive. For an electrical detector, the signal current is plotted as a function of the potential difference (voltage) applied to the operating electrode with respect to the concentration of analyte provided. The resulting sigmoidal graph is termed the current-voltage curve or hydrodynamic voltammogram (HDV). HDV allows the best choice of potential difference applied to the operating electrode to allow maximizing the observed signal. An important advantage of ECD is inherent sensitivity with current levels of detection in the subfemtomolar range.
Many chemicals and compounds are electrochemically active, including many biochemicals, drugs, and herbicides. Compounds released by the chromatograph can be decomposed effectively even when their half-wave potential difference (potential difference at half of the signal maximum) is only 30 to 60 mV different.
The recently developed electrical sensors provide sensitivity, identity verification and degradation of the coexistent compounds when used as detectors in liquid chromatographic separations. Thus, the detectors thus arranged add another set of separations that accompany the detector itself. The current apparatus in principle has 16 channels limited only by the rate at which data can be obtained. The number of compounds that can be degraded on the EC array is defined by chromatography (i. E., Defined plate count). However, if the two or more compounds shipped by chromatography have deviations at a half-wave potential difference of 30 to 60 mV, the arrangement can distinguish the compounds. The performance of the compounds, which may be electrochemically active, depends on the retention of the EC activation groups (i.e., -OH, -O, -N, -S).
Compounds that are successfully detected using an electrical detector include 5-hydroxytryptamine, 3-methoxy-4-hydroxyphenyl-glycol, homogentisic acid, dopamine, methaneprine, 3- Phenol, o-cresol, pyrogallol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 4-hydroxytryptophan, 2-nitrophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 2,4,5-trichlorophenol, 4-chloro-3-methylphenol, 4-hydroxyaniline, 1,2-phenylenediamine, benzocatechin, buturon, chlorotoluron, dioron, isoproturon Methionine, tryptophan, tyrosine, 4-aminobenzoic acid, 4-hydroxybenzoic acid, 4-hydroxycoumaric acid, 7-hydroxycoumaric acid, - epoxides such as methoxycoumarin, apigenin baicalein, caffeic acid, catechin, cetaine, chlorogenic acid, daidzein, diticetin, diosmetin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, , Erythritol, erupatrin, ferulic acid, phygetin, gallantin, galactan, gardanine, genistein, gentisic acid, hesperidin, irigenin, caneferol, ricodianidin, luteolin, mangosteen, Naringin, Naryutin, Pelargendin, Phenonidine, Floretin, Pranenegin, Protocatechuic acid, Raminetin, Quercetin, Sakuranetin, Scutelraine, Scopoletin, Sealdehyde, Sicric acid, Ricinolein, N-methyl-r-salicolinol, tetrahydroisoquinoline, benzoic acid, benzoic acid, benzoic acid, , Amitriptyline, apomorphine, capsaicin, clodia Dexamethasone, morphine, morphine-3-glucuronide, nortriptyline, morpholine-3-glucoside, dexamethasone, dexamethasone, Dihydroxybenzylamine, 3,4-dihydroxymandelic acid (DOMA), 3,4-dihydroxybenzaldehyde, oxalic acid, (DOPAC), 3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylglycol (DHPG), 3-hydroxyanthranilic acid, 2-hydroxyphenylacetic acid Hydroxybenzoic acid (4HBAC), 5-hydroxyindole-3-acetic acid (5HIAA), 3-hydroxycinolenine, 3-hydroxymandelic acid, 3-hydroxy- -Hydroxyphenylacetic acid (4HPAC), 4-hydroxyphenyllactic acid (4HPLA), 5-hydroxytryptophan (5HTP), 5-hydroxytryptophol (5HT), 5-hydroxytryptamine sulfate, 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-methoxytryptamine, 5-methoxytryptophan, Dopamine, 3-methylcysteine, 3-methylguanine, vapotenin, dopamine, dopamine-3-glucuronide, dopamine-3-sulfate, dopamine (HVA), homovanillyl alcohol (HVOL), homobarathic acid (HVA), benzoic acid, and the like. N-methyltripamine, N, N-dimethyltryptamine, N, N-dimethyltrimethamine, N, N-dimethyltrimethamine, indole-3-acetic acid, indole-3-lactic acid, quinoline, melatonin, (Vma), xanthine (vma), xanthine, xanthine, xanthine, xanthine, xanthine, And xanthosine. Other suitable compounds are described, for example, in Jane, I., et al. J. Chrom. 323: 191-225 (1985) and Musch, G., et al., J. Chrom. 348: 97-110 (1985). Such compounds may be incorporated into compounds of formula T-L-X by methods known in the art. For example, a compound having a carboxylic acid group can react with an amine, a hydroxyl, and the like to form an amide, an ester, and other bonds between T and L.
In addition to the above characteristics and regardless of the intended detection method, a tag having a chemical structure of the module is preferred. This aids in the establishment of a number of structurally related taches, using techniques of compounding chemistry. For example, the T ^ms group preferably has some characteristics. Preferably, a residue containing T ^ms contains a functional group that supports a single ionized charge state when mass spectrometric analysis (more simply referred to as " mass spectral sensitivity enhancer " group or MSSE). It can also be used as a member of one of the residue groups, preferably containing T ^ms , if the mass / charge ratios of the respective members in the group are different but have approximately the same sensitivity in a mass spectrometer. Thus, multiple groups have the same MSSE. It has been found convenient to allow the tack element itself to appear as containing a module by forming a tack reactant through a synthesis scheme of the module to allow the formation of groups of compounds.
In the case of a preferred access module of the structure of the T ^ms group, T ^ms groups are as follows: Formula 6:
T ² - (JT ³ -) _n -
In the above formula
T ² is an organic residue formed from carbon and at least one of hydrogen, fluorine, iodine, oxygen, nitrogen, sulfur and phosphorus with a mass range of 15 to 500 dalton,
T ³ is an organic residue formed from carbon and at least one of hydrogen, fluorine, iodine, oxygen, nitrogen, sulfur and phosphorus with a mass range of 50 to 1000 dalton,
J is a direct bond or a direct bond or an amide, ester, amine, sulfide, ether, thioester, disulfide, thioether, urea, thiourea, carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine, oxime, hydrazone , A functional group such as a phosphate, a phosphonate, a phosphoramid, a phosphonamide, a sulfonate, a sulfonamide or a carbon-carbon bond,
n is an integer ranging from 1 to 50 such that each of T ³ and J is independently selected when n is greater than one.
The structure of the module T ² - (JT ³ -) _n - provides a convenient entry for a group of TLXs, where each member of the group has a different T group. For example, if T is T ^ms , and each group member preferably has the same MSSE, one of the T ³ groups may provide an MSSE structure. To provide diversity among the members of the group with respect to the mass of T ^ms , the T ² group may vary among the group members. For example, one group member may be T ² methyl, while another member may be T ² ethyl and another member T ² may be a profile or the like.
To provide " large " or large leaps in mass, the T ³ group can be designed to add significant (e.g., hundreds or hundreds) mass units to the TLX. Such T ³ groups may be referred to as molecular weight range modifier groups (" WRA "). WRA is very useful when one works with a single set of T < ^{2 >} groups, and the mass can be extended over a limited range. A single set of T ² groups can be used to form a T ^ms group with a broad mass range simply by incorporating one or more WRA T ³ groups into the T ^ms . Thus, using a simple example, if the mass range for a T ^ms of a set of T ² groups is 250-340 daltons, then by adding a single WRA with, for example, 100 dalton as a T ³ group, the same set of T ² groups Lt; RTI ID = 0.0 > daltons < / RTI > during use. Similarly, by applying a two MWA groups of 100dalton (as a T ³ group, respectively) provide for access to the mass range of 450 to 540dalton and, at this time continue to very large for the T ^ms group by the application of this WRA groups progressively Lt; RTI ID = 0.0 > mass range. ≪ / RTI > Formula T ² - (JT ³ -) _n -LX preferred compounds of the formula _{R VWC - (R WRA) w} -R MSSE -LX ( here, Group VWC is a "T ^2", each of the WRA and MSSE groups are " T ³ " group). This structure is shown in Figure 12 and represents one module approach to the fabrication of T ^ms .
In the formula T ² - (JT ³ -) _n -, T ² and T ³ are preferably selected from hydrocarbyl, hydrocarbyl-O-hydrocarbylene, hydrocarbyl-S-hydrocarbylene, hydrocarbyl- (Hydrocarbyl) hydrocarbyl, hydrocarbyl acyl-hydrocarbyl, heterocyclyl hydrocarbyl [wherein the hydrocarbyl is selected from the group consisting of hydrocarbyl, carbene, hydrocarbyl-amide- , Wherein the heteroatom (s) is selected from oxygen, nitrogen, sulfur and phosphorus, the substituents being selected from the group consisting of hydrocarbyl, (Hydrocarbyl) hydrocarbylene, N, N-di (hydrocarbyl) hydrocarbylene and hydrocarbyl-O-hydrocarbylene, hydrocarbyl-N-hydrocarbylene, hydrocarbyl- Hydrocarbyl acyl-hydrocarbyl Lt; / RTI > In addition, T ² and / or T ³ may be any derivative of the T ² / T ³ group listed above so that at least one hydrogen is replaced by fluorine.
Also with respect to the formula T ² - (JT ³ -) _n -, preferred T ³ is a group of the formula -G (R ² ) -, wherein G is a C _1-6 alkylene chain having a single R ² substituent . Thus, G is ethylene (-CH ₂ -CH ₂ -) in the case, the two ethylene any one of the carbons may have a R ² substituent, R ² is alkyl, alkenyl, alkynyl, cycloalkyl, aryl- Substituted fused alkyl, fused cycloalkyl, cycloalkenyl, aryl, aralkyl, aryl-substituted alkenyl or alkynyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted cycloalkyl, biaryl, alkoxy, alkenoxy, Aryl-substituted alkenyloxy or alkynyloxy, aryl-substituted alkenyloxy or alkynoxy, aryloxy-substituted alkenyloxy or alkynyloxy, aryloxy- Substituted alkyl, alkylcarbonylamino-substituted alkyl, aminocarbonyl-substituted alkyl, heterocyclyl, heterocyclyl-substituted alkyl < RTI ID = 0.0 > , Heterocyclyl-substituted amino, carboxyalkyl-substituted Aralkyl, oxo carbocyclyl-fused aryl and heterocyclylalkyl; Substituted alkyl, alkoxy-substituted alkyl, aralkoxy-substituted alkyl, amino-substituted alkyl, alkoxy-substituted alkyl, aralkoxy-substituted alkyl, Substituted alkyl, thiol-substituted alkyl, monoalkylsulfonyl-substituted alkyl, (hydroxy-substituted alkylthio) -substituted alkyl, thioalkoxy- Substituted alkyl, hydrocarbyl acylamino-substituted alkyl, heterocyclylamino-substituted alkyl, hydrocarbyl-substituted heterocyclylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl, arylsulfonyl Substituted or unsubstituted alkyl, amino-substituted alkyl, morpholino-alkyl, thiomorpholino-alkyl, morpholinocarbonyl-substituted alkyl, thiomorpholinocarbonyl- Or N, N- [dialkyl, dialkenyl, dialkynyl or (Al , Alkenyl) -amino] carbonyl-substituted alkyl, heterocyclylaminocarbonyl, heterocyclylalkyleneaminocarbonyl, heterocyclylaminocarbonyl-substituted alkyl, heterocyclylalkyleneaminocarbonyl- Substituted alkyl, substituted alkyl, substituted alkyl, N, N- [dialkyl] alkylenaminocarbonyl, N, N- [dialkyl] alkylenaminocarbonyl-substituted alkyl, alkyl-substituted heterocyclylcarbonyl, S-methylcysteine, methionine and the corresponding sulfoxides and sulfone derivatives thereof, glycine, leucine, glycine, glycine, Butyrine, threonine, serine, aspartic acid, beta-cyanoalanine, and allotreonine, such as isoleucine, isoleucine, allo-isoleucine, tert-leucine, norleucine, phenylalanine, tyrosine, tryptophan, proline, alanine, ornithine, histidine, ; Mono-or diarylaminocarbonyl, alkylarylaminocarbonyl, diarylaminocarbonyl, mono-or diarylcarbonyl, mono- or dialkylaminocarbonyl, Mono- or dialkylamino, mono-or diarylamino, alkylarylamino, diarylamino, mono- or diacylamino, mono- or diacylamino, Is selected from amino acid side chains selected from alkyl substituted by a substituent selected from alkoxy, alkenoxy, aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy and heterocyclyl.
Preferred compounds of the formula T ² - (JT ³ -) _n -LX are as follows:
In this formula,
G is hydrogen in the illustrated one and only one of the CH ₂ groups by a single "G" -, and (CH ₂₎ to be replaced by an amide ^{_{-T 4 1-6, - (CH 2}} ) c
T ² and T ⁴ are organic moieties of the formula C _1-25 N _0-9 O _0-9 H _α F _{β which} make the sum of α and β sufficient to satisfy unsatisfactory C, N and O atoms of valence ego,
Amide ego,
R ¹ is hydrogen or C _1-10 alkyl,
c is an integer of 0 to 4,
n is an integer of 1 to 50, and when n is greater than 1, G, c, amide, R ¹ and T ⁴ are each independently selected.
In a further preferred embodiment, the compound of formula T ² - (JT ³ -) _n -LX is as in formula (8)
In this formula,
T ⁵ is an organic moiety of the formula C _1-25 N _0-9 O _0-9 H _α F _β which makes the sum of α and β sufficient to satisfy unsatisfactory valence C, N and O atoms,
T ⁵ comprises a tertiary or quaternary amine or organic acid,
m is an integer from 0 to 49,
T ^2, T ⁴ , R ¹ , L and X are as defined above.
Another preferred compound having the formula T ² - (JT ³ -) _n -LX is as follows:
In this formula,
T ⁵ is an organic moiety of the formula C _1-25 N _0-9 O _0-9 H _α F _β which makes the sum of α and β sufficient to satisfy unsatisfactory valence C, N and O atoms,
T ⁵ comprises a tertiary or quaternary amine or organic acid,
m is an integer from 0 to 49,
T ^2, T ⁴ , c, R ¹ , "amide", L and X are as defined above.
In the above structure with the T ⁵ group, -amide-T ⁵ is preferably one which is conveniently prepared by reacting an organic acid with a free amino group extending from G:

When the above compound has a T ⁵ group, the "G" group has the following free-carboxylate group, which has a free carboxyl group (or its equivalent) and is conveniently prepared by reacting a suitable organic amine with a free carboxyl group extending from G The -T ⁵ group is preferred:

In three preferred embodiments of the present invention, the structure of T-L-MOI is as follows:
In this formula,
T ² and T ⁴ are such that the sum of α, β and δ is sufficient to satisfy unsaturated C, N, O, S and P atoms of valence C _1-25 N _0-9 O _0-9 S _0-3 P _0-3 H F I
G is one and only one hydrogen in the CH ₂ groups represented by each G-amide and (CH _{2) 1-6} is replaced by -T ^4, - (CH _{2) C}
Amide ego,
R ¹ is hydrogen or C _1-10 alkyl,
c is an integer of 0 to 4,
"C ₂ -C ₁₀ " is a hydrocarbylene group having 2 to 10 carbon atoms,
"ODN-3'-OH" represents a nucleic acid fragment having a terminal 3 'hydroxy group (that is, a nucleic acid fragment bound to (C ₁ -C ₁₀ ) at a terminal other than 3' of the nucleic acid fragment)
n is an integer of 1 to 50, and when n is greater than 1, G, c, amide, R ¹ and T ⁴ are each independently selected. Preferably there are no three heteroatoms bound to a single carbon atom.
In the structure as shown above containing a T ² -C (═O) -N (R ¹ ) - group, this group represents only the amine of HM (R ¹ ) - for example, With an organic acid selected from the group consisting of formic acid, acetic acid, propiolic acid, propionic acid, fluoroacetic acid, 2-butenoic acid, cyclopropanecarboxylic acid, butyric acid, methoxyacetic acid, difluoroacetic acid, 4-pentenoic acid, cyclobutanecarboxylic acid, 3,3-dimethyl acrylic acid, valeric acid, N, N-dimethylglycine, N-formylgly-OH, ethoxyacetic acid, (methylthio) acetic acid, Acetic acid, hexanoic acid, Ac-gly-OH, 2-hydroxy-2-methylbutyric acid, benzoic acid, nicotinic acid, 2- Pyrazinecarboxylic acid, 1-methyl-2-pyrrolecarboxylic acid, 2-cyclopentane Acetic acid, cyclopentylacetic acid, (S) - (-) - 2-pyrrolidone-5-carboxylic acid, N-methyl-L-proline, heptanoic acid, Ac- 2-hydroxybutyralic acid, 2- (2-methoxyethoxy) acetic acid, p-toluic acid, 6-methylnicotinic acid, , 4-fluorobenzoic acid, 3,5-dimethylisoxazole-4-carboxylic acid, 3-cyclopentylpropionic acid, octanoic acid, N, N-dimethylsuccinic acid, phenylpropiolic acid, cinnamic acid, 3-fluoro-4-methylbenzoic acid, Ac-DL-propargylglycine, 3- (trifluoromethyl) butyric acid, 1-piperidine 2-carboxylic acid, 2-benzofuranocarboxylic acid, benzotriazole-5-carboxylic acid, 4-n-propyl acid, N-acetylproline, Benzoic acid, 3-dimethylaminobenzoic acid, 4-ethoxybenzoic acid, 4- (methylthio) benzoic acid, N- (2-furoyl) 4-methoxybenzoic acid, Tfa-Gly-OH, 2-naphthoic acid, quinald acid, Ac-L-Ile-OH, 3- methylidene- Methyl-indole-2-carboxylic acid, 2,3,6-trifluorobenzoic acid, N-formyl-L-Met-OH, 2- [2- (2-methoxyethoxy) ethoxy] N-benzoylglycine, 5-fluoroindole-2-carboxylic acid, 4-n-propoxybenzoic acid, 4-acetyl-3,5-dimethyl-2-pyrrolecarboxylic acid, 3,5 2-naphthylacetic acid, 4- (1H-pyrrol-1-yl) benzoic acid, indole-3-propionic acid, m-trifluoromethylbenzoic acid 2-carboxylic acid, 4-pentylbenzoic acid, Bz-b-Ala-OH, 4-diethylaminobenzoic acid, 4-n-butoxybenzoic acid, 3- 4-biphenylcarboxylic acid pivaloyl-Pro-OH, octanoyl-Gly-OH, (2-naphthoxy) acetic acid, indole-3-butyric acid, 4 -Acetic acid, 4- (trifluoromethoxy) benzoic acid, Ac-L-Phe-OH, 4-pentyloxybenzoic acid, Z-Gly-OH, 4-carboxy -N- (greener-2-ylmethyl) pyrrolidin-2-one, 3,4-diethoxy-benzoic acid, 2,4-dimethyl -5-CO ₂ Et- pyrrole-3-carboxylic acid, N- ( Phenoxyphenyl) succinic acid, 3,4,5-trimethoxybenzoic acid, N-phenylanthranilic acid, 3-phenoxybenzoic acid, nonanoyl-Gly-OH, 2-phenoxypyridine- (5-methyl-2-phenyloxazol-4-yl) acetic acid, 4- (2- Benzoic acid, 5-methoxy-2-methylindole-3-acetic acid, trans-4- 4-dimethoxyphenyl) butyric acid, Ac-o-fluoro-DL-Phe-OH, N- (4-fluorophenyl) glutaric acid, 4 '-Ethyl-4-biphenylcarboxylic acid, 1,2,3,4-tetra N-decanoyl-Gly-OH, (+) - 6-methoxy-a-methyl-isoquinolinecarboxylic acid, 2-naphthaleneacetic acid, 3- (trifluoromethoxy) cinnamic acid, N-formyl-DL-Trp-OH, (R) - (+) - a- methoxy-a- (trifluoromethyl) , Bz-DL-Leu-OH, 4- (trifluoromethoxy) phenoxyacetic acid, 4-heptyloxybenzoic acid, 2,3,4-trimethoxycinnamic acid, 2,6-dimethoxybenzoyl- (2,4,5-trimethoxyphenyl) propionic acid, 2,3,4,5,6-pentafluorophenoxyacetic acid, N- (2,4-difluorophenyl) glutaric acid, N (4-fluorobenzoyl) benzoic acid, 5-trifluoromethoxyindole-2-carboxylic acid, N- (2,4-difluorophenyl) diglycolic acid, Ac- L-Trp-OH, Tfa-L-phenylglycine-OH, 3-indenylbenzoic acid, 3- (4-n-pentylbenzoyl) propionic acid, 2- -Met-OH, 3,4,5-triethoxybenzoic acid, N- 3-iodo-4-methylbenzoic acid, 3,5-bis (trifluoromethyl) benzoic acid, Ac-5-methyl-DL-TrP- 4-n-hexylbenzoyl) propionic acid, N-hexanoyl-L-Phe-OH, 4-nonyloxybenzoic acid, 4 '- (trifluoromethyl) (4-n-heptylbenzoyl) propionic acid, N-heptanoyl-L-Phe-OH, 4- (2-hydroxyhexafluoroisopropyl) benzoic acid, N-myristoyl-Gly-O-benzoic acid, N- OH, 3- (4-n-octylbenzoyl) propionic acid, N-octanoyl-L-Phe-OH, 4-undecyloxybenzoic acid, 3- (3,4,5-trimethoxyphenyl) propionyl- OH, 8-iodonaphthoic acid, N-pentadecanoyl-Gly-OH, 4-dodecyloxybenzoic acid, N-palmitoyl-Gly-OH and N-stearoyl-Gly-OH. These organic acids may be obtained from one or more manufacturers (Advanced ChemTech, Louisville, KY; Bachem Bioscience Inc., Torrance, CA; Calbiochem-Novabiochem Corp., San Diego, Calif .; Farchan Laboratories Inc., Gainesville FL; Lancaster Synthesis, Windgam NH; and MayBridge Chemical Company (c / o Ryan Scientific), Columbia, SC. The catalogs from these manufacturers use the abbreviations used above to define the acid.
f. Combination chemistry as a means for preparing tack
Combination chemistry is one form of synthetic method that leads to the production of a number of chemicals (for example, International Patent Application No. WO 94/08051). Such a combination material can be used as a tag for identification of the molecule of interest (MOI). Combination chemistry can be defined as a global and repeated covalent association of a set of different " building blocks " of a varying structure with respect to each other to form a large array of reverse molecular sieves. The building block may take many forms of both natural and synthetic, such as nucleophiles, electrophiles, dienes, alkylating or acylating agents, diamines, nucleotides, amino acids, sugars, lipids, organic monomers, synthons and combinations thereof . The chemical reactions used to link the building blocks may include alkylation, acylation, oxidation, reduction, hydrolysis, substitution, removal, addition, ring closure, condensation, and the like. By such a process, oligomeric, non-oligomeric, or compound materials containing them can be prepared. When oligomeric, the compound may be branched, non-branched, or cyclic. Examples of oligomeric structures that can be prepared by the combination method include oligopeptides such as oligopeptides, oligonucleotides, oligosaccharides, polyolipids, polyesters, polyamides, polyurethanes, polyureas, polyethers, poly (phosphorus derivatives) (Sulfur derivatives) such as sulfones, sulfonates, sulfites, sulfonamides, sulfenamides, and the like, as well as phosphates, phosphates, phosphates, phosphonates, phosphoamides, phosphonamides, phosphites, .
One common form of oligomeric associative material is a peptide combination material. With recent advances in peptide chemistry and molecular biology, it has become possible to manufacture and use from 10 to 1 million different peptide sequences. These materials can be classified into three broad categories. One category of materials involves the chemical synthesis of soluble, non-ligand-bound peptide materials (Houghten et al., Nature 354: 84, 1991). The second category involves the chemical synthesis of support-bound peptide materials present on solid supports such as plastic pins, resin beads, or facets (Geysen et al., Mol. Immunol. 23: 709,1986; Lam et al., Nature 354: 82, 1991; p Eichler and Houghten, Biochemistry 32: 11035, 1993). In these first two categories, building blocks are typically L-amino acids, D-amino acids, unnatural amino acids, or some mixture or combination thereof. The third category uses molecular biology to produce peptides or proteins on the surface of filamentous phage particles or plasmids (Scott and Craig, Curr. Opinion Biotech. 5: 40, 1994). Soluble, non-lipid-coupled peptide materials appear to be suitable for a number of applications, including as a tack. A list of commercially available chemical induction forms in peptide materials can be amplified by steps such as hypermethylation (Ostresh et al., Proc. Natl. Acad. Sci., USA 91: 11138,1994).
Many modifications of the peptide conjugate material can modify the peptide backbone and / or the amide bond is replaced by a similar group. Amide-like groups that may be used include urea, urethane, and carbonylmethylene groups. By a rearrangement of the backbone such that the side chains occur from amide nitrogen of each amino acid rather than alpha-carbon (Simon et al., Proc. Natl. Acad. Sci., USA 89: 9367, 1992).
Another common form of oligomeric associative material is that the building block can be a natural or synthetic nucleotide or a mixture of oligonucleotides, including where various organic and inorganic groups can replace the phosphate linkage and where nitrogen or sulfur can replace oxygen in the ether linkage. In the case of some forms of polysaccharide derivatives, it is an oligonucleotide combination material (Schneider et al., Biochem. 34: 9599,1995; Freier et al., J. Med. Chem. 38: 344, 1995; Frank, J. Biotechnology 41: 259, 1995; Schneider et al., Published PCT WO 942052; Ecker et al., Nucleic Acids Res. 21: 1853,1993).
More recently, collection-combining products of non-oligomeric, low molecular weight compounds have been described (DeWitt et al., Proc. Natl. Acad. Sci., USA 90: 690, 1993; Bunin et al., Proc. Natl. Acad. Sci., USA 91: 4708,1994). Suitable structures for the synthesis of low molecular weight materials include a wide range of organic molecules such as heterocycle, aromatic, Catalysts as well as combinations thereof.
g. Concrete method for combination synthesis of tack
Two methods for the preparation and use of various sets of MS tack containing amines are outlined below. In both methods, solid-phase synthesis can be used to achieve co-parallel synthesis of a number of linkers that are tacked using techniques of combinatorial chemistry. In the first method, cleavage of the tangs from the oligonucleotide ultimately results in the separation of the carboxylamide. In the second method, the carboxylic acid is prepared by truncating the tack. The chemical components and linker elements used in this method use the following abbreviations:
R resin
FMOC Fluorenylmethoxycarbonyl protective group
All ally protection group
CO ₂ H carboxylic acid group
CONH ₂ carboxylic acid amide group
NH ₂ amino group
OH hydroxyl group
CONH amide bond
COO ester bond
NH ₂ -Rink-CO ₂ H 4 - [( -Amino) -2,4-dimethoxybenzyl] -phenoxybutyric acid
(Rink linker)
OH-1MeO-CO ₂ H (4-hydroxymethyl) phenoxybutyric acid
OH-2MeO-CO ₂ H (4-hydroxymethyl-3-methoxy) phenoxyacetic acid
Amino acids having aliphatic or aromatic amine functional groups on the NH ₂ -A-COOH side chain
X1 ..... Xn-COOH A set of n different carboxylic acids having intrinsic molecular weights
(N) oligonucleotides < RTI ID = 0.0 >
HBTU O-Benzotriazol-1-yl-N, N, N ', N', - tetramethyluronium
Hexafluorophosphate
The order of the steps in the method 1 is as follows:
OH-2MeO-CONH-R
↓ FMOC-NH-Rink-CO ₂ H; Coupling (e. G., HBTU)
FMOC-NH-Rink-COO-2MeO-CONH-R
↓ piperidine (FMOC removal)
MH ₂ -Rink-COO-2MeO-CONH-R
↓ FMOC-NH-A-COOH; Coupling (e. G., HBTU)
FMOC-NH-A-CONH-Rink-COO-2MeO-CONH-R
↓ piperidine (FMOC removal)
MH ₂ -A-CONH-Rink-COO-2MeO-CONH-R
↓ Split into n aliquots
↓ ↓ ↓ ↓ ↓ Coupling to n different acids X1 .... Xn-COOH
X1... Xn-CONH-A-CONH-Rink-COO-2MeO-CONH-R
↓ ↓ ↓ ↓ ↓ Cutting of the linker tapped from resin with 1% TFA
X1 .... Xn-CONH-A- CONH-Rink-CO 2 H
↓ ↓ ↓ ↓ ↓ Coupling to n raising (raising 1 ... raising (n))
(E. G., Via Pfp esters)
X1 .... Xn-CONH-A-CONH-Rink-CONH-Oligo 1 ... Oligo (n)
Collection of oligo set with tack ↓
↓ Perform sequence analysis
↓ Separation of fragments of different lengths from sequencing reactions
(E. G., Via HPLC or CE)
↓ Cutting tacks from linker with 25-100% TFA
X1 .... Xn-CONH-A-CONH
↓
Analysis by mass spectrometry
The order of steps in method 2 is as follows:
OH-1MeO-CO ₂ -All
FMOC-NH-A-CO ₂ H; Coupling (e. G., HBTU)
FMOC-NH-A-COO-1MeO-CO ₂ -Al
↓ Palladium (allyl removal)
FMOC-NH-A-COO-1MeO-CO ₂ H
OH-2MeO-CONH-R; Coupling (e. G., HBTU)
FMOC-NH-A-COO-1MeO-COO-2MeO-CONH-R
↓ piperidine (FMOC removal)
NH ₂ -A-COO-1MeO-COO-2MeO-CONH-R
↓ Split into n aliquots
↓ ↓ ↓ ↓ ↓ Coupling to n different acids X1 .... Xn-CO ₂ H
X1 .... Xn-CONH-A-COO-1MeO-COO-2MeO-CONH-R
↓ ↓ ↓ ↓ ↓ Cutting of the linker tapped from resin with 1% TFA
X1 .... Xn-CONH-A- COO-1MeO-CO 2 H
↓ ↓ ↓ ↓ ↓ Coupling to n raising (raising 1 ... raising (n))
(E. G., Via Pfp esters)
X1 .... Xn-CONH-A-COO-1MeO-CONH-oligo 1 ... oligo (n)
Collection of oligo set with tack ↓
↓ Perform sequence analysis
↓ Separation of fragments of different lengths from sequencing reactions
(E. G., Via HPLC or CE)
↓ Cutting tacks from linker with 25-100% TFA
X1 .... Xn-CONH-A- CO 2 H
↓
Analysis by mass spectrometry
2. Linker
As used herein, a "linker" component (or L) is an organic chemical that is used to directly bond a covalent bond or "tack" (or T) to a "molecule of interest" (or MOI) Group. In addition, the direct bond itself or one or more bonds in the binding component can be cleaved under conditions that allow T to be released (in other words, cleaved) from the residue of the T-L-X compound (including the MOI component). Tack strain components present in T must be stable to the cutting conditions. Preferably, the cleavage can be performed rapidly within a few minutes and within about 15 seconds.
Typically, the linker is used to link the tag of each large set to the MOI of each similar large set. Typically, a single tack-linker combination is attached to each MOI (in order to provide various TL-MOIs), but in some cases one or more tack-linker combinations may be attached to each individual MOI ) to provide n-MOI). In another embodiment of the present invention, two or more tacks are coupled to a single linker through a plurality of independent sites in the linker, and the plurality of tack-linker combinations are coupled to separate MOIs (various (T) to provide nL-MOI).
After various manipulations of the tapped MOI set, the tack is separated from the MOI by breaking one or more covalent bonds in the linker using specific chemical and / or physical conditions. The cleavable bond (s) may or may not be part of the same bond formed when the tacky, linker and MOI are linked together. The design of the linker will in many cases determine the conditions that can be cut. Therefore, the linker can identify the congestion by a condition that can be specifically accommodated. If the linker is photolabile (i. ^E. Tends to be cleaved by exposure to radiation), the linker can be denoted by L ^hv . Similarly, L ^acid , L ^base , L ^[O] , L ^[R] , L ^enz , L ^elc , L ^Δ and L ^ss are acid, base, chemical oxidation, chemical reduction, Quot; catalyst "), electrochemical oxidation or reduction, elevated temperature (" thermal "), and cleavage by thiol exchange.
Certain types of linkers are unstable to a single type of cleavage condition while others are unstable to some form of cleavage conditions. Also, in a linker capable of combining multiple tacks (to provide a structure in the form of (T) n-L-MOI), each tack-binding site may be unstable to different cutting conditions. For example, in a linker with two tacks attached to a linker, one of the links may be only unstable to the base, and the other may be only unstable to photodegradation.
Linkers useful in the present invention have several advantages:
1) The linker has a chemical handle (L _h ) that can be attached to the MOI.
2) The linker has a second separate chemical handle (L _h ) attaching the tack to the linker. When multiple tags are attached to a single linker ((T) nL-MOI type structure), individual handles are present in each tack.
3) The linker is stable for all operations performed except that the cleavage is allowed and the residue containing T is released from the residue of the compound containing MOI. Thus, the linker is stable during attachment of the linker to the MOI while it is attached to the linker, and is stable during operation of any MOI while attaching the tackle and linker (T-L) thereto.
4) The linker does not significantly interfere with the operations performed on the MOI while the T-L is attached. For example, when T-L is attached to an oligonucleotide, T-L should not significantly interfere with any hybridization or enzymatic reaction (e.g., PCR) performed on the oligonucleotide. Similarly, when T-L is attached to an antibody, it should not significantly interfere with antigen recognition by the antibody.
5) The tack from the residue of the compound is cleaved in a highly controlled manner using physical or chemical processes that do not adversely affect the detection performance of the tack.
For any given linker, it is preferred that the linker can be attached to various MOIs, and various tacks can be attached to the linker. This flexibility is advantageous since once it is prepared it allows the T-L conjugate to be used with several different sets of MOIs.
As illustrated above, the preferred linker is represented by Formula 5:
Formula 5
L _h -L ¹ -L ² -L ³ -L _h
In this formula,
Each L _h is a reactive handle that can be used to link the linker to the chemical reactants and molecular reactants of interest,
L ² is an essential part of the linker because it imparts instability to the linker,
L ¹ and L ³ are any group effectively used to separate L ² from the handle L _h .
L ¹ (closer to T than L ³ by definition) is used to separate T from the required instability residue L ² . This separation may be useful when the cleavage reaction forms a particular reactive entity (e. G., A free radical) that can cause an irregular change in the structure of the T containing moiety. As the cleavage site is further separated from the residue containing T, the possibility that the reactive substance formed at the cleavage site can disrupt the structure of the residue containing T is reduced. Also, since the atom in Ll typically can be in a residue containing T, the Ll atom can impart desired properties to the residue containing T. For example, when the residue containing T is a residue containing T ^ms and when the blocked amine is preferably present as a part of the structure of the residue containing T ^ms (for example, to be used as MSSE ), The interrupted amine may be present in the L ¹ labile moiety.
In other cases, L < ^{1 >} and / or L < ³ > may be present in the linker simply because the market maker of the linker chooses to market the linker in the form of L ¹ and / or L ³ groups. In this case, in using linkers having L ¹ and / or L ³ groups, even if such groups can not impart any particular performance advantages to compounds incorporated therein (as long as they do not inhibit the cleavage reaction) It is not harmful though. Thus, the present invention allows for L < ^{1 >} and / or L < ³ > groups to be present in the linker.
The L < ^{1 >} and / or L < ³ > groups may be linked directly (in this case the group is not effectively present), a hydrocarbylene group (e.g. alkylene, arylene, cycloalkylene, etc.) car bilren of (here, w is from 1 to about 10 _{(e.g., -O-CH 2 -, -O} -CH 2 CH (CH) - , etc.) or a side Rocca bilren - (O- dihydro car bilren) _w (E.g., -CH ₂ -O-Ar-, -CH ₂ - (O-CH ₂ -CH ₂ ) ₄ -, etc.).
With the advent of solid phase synthesis, a number of articles have been published relating to linkers that are unstable to certain reaction conditions. In a typical solid phase synthesis, the solid support binds to the reactive sites via the unstable linker, and the molecules to be synthesized are formed at the reactive sites. When the molecule is fully synthesized, the structure of the solid support-linker-molecule is placed in a cleavage condition that releases the molecule from the solid support. Unstable linkers developed for use in this document (or which can be used in these documents) can be used rapidly as linker reagents in the present invention.
Lloyd-Williams, P., et al., &Quot; Convergent Solid-Phase Peptide Synthesis ", Tetrahedron Report No. 2 347, 49 (48): 11065-11133 (1993) provide various discussions of linkers that are unstable to radiation (e.g., photodegradation) as well as acid, base and other cleavage conditions. Additional sources of information on unstable linkers are readily available.
As described above, different linker forms can impart cutting performance (" unstable performance ") under different specific physical or chemical conditions. Examples of conditions used for cleaving linkers of various designs include acid, base, oxidation, reduction, fluorine, thiol exchange, photolysis, and conditions by enzymes.
Examples of cleavable linkers that meet the general standard for the linkers listed above are those well known to those skilled in the art and include those shown in the catalog available from Pierce, Rockford, IL. Examples of these include:
· Ethylene glycobis (succinimidyl stearate) (EGS), an amine reactive cross-linking agent that can be cleaved by hydroxylamine (1M at 37 ° C for 3 to 6 hours);
· Amine reactive crosslinking agents which can be cleaved by disuccinimidyl tartrate (DST) and sulfo-DST, 0.015 M sodium periodate
Amine-reactive crosslinking agents which can be cleaved by bis [2- (succinimidyloxycarbonyloxy) ethyl] sulfone (BSOCOES) and sulfo-BSOCOES, base (pH 11.6);
A pyridyldithiol cross-linking agent which can be cleaved by 1,4-di- [3 '- (2'-pyridyldithio (propionamido)) butane (DPDPB), thiol exchange or reduction;
- Pyridyl dithiols that can be cleaved by thionylation or reduction, such as N- [4- (p-azidosalicylamido) -butyl] -3 '- (2'-pyridyldithio An organic crosslinking agent;
Photoreactive crosslinking agents which can be cleaved by bis- [beta-4- (isoindolyl salicylamido) -ethyl] disulfide, thiol exchange or reduction;
Photoreactive crosslinking agents which can be cleaved by N-succinimidyl- (4-azidophenyl) -1,3'-dithiophosphonate (SADP), thiol exchange or reduction;
-Sulphosuccinimidyl-2- (7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3'-dithiophosphonate (SAED), cut by thiol exchange or reduction Photoreactive cross-linking agents;
- Sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido) -ethyl-1,3'-dithiophosphonate (SAND), light which can be cleaved by thiol exchange or reduction Reactive crosslinking agent.
Other examples of linkers that can be cut and the cutting conditions that can be used to release the tack are as follows. The silyl bonding groups can be cleaved by fluorine or under acidic conditions. The 3-, 4-, 5- or 6-substituted 2-nitrobenzyloxy or 2-, 3-, 5- or 6-substituted 4-nitrobenzyloxy bond group is cleaved by light source . 3-, 4-, 5-, or 6-substituted-2-alkoxyphenoxy or 2-, 3-, 5- or 6-substituted-4-alkoxyphenoxy bond group Ce (NH _{4) 2} (NO ₃ ) ₆ (oxidation). NCO ₂ (urethane) linkers can be cleaved by hydroxides (bases), acids or LiAlH ₄ (reduction). The 3-pentenyl, 2-butenyl or 1-butenyl bond group can be cleaved by O ₃ , O _s O ₄ / IO ₄ ^- , or KMnO ₄ (oxidation). The 2- [3-, 4- or 5-substituted-furyl] oxy bond group can be cleaved by O ₂ , Br ₂ , MeOH or an acid.
Other labile conditions for cleaving bond groups include: t-alkyloxy bond groups can be cleaved by an acid; Methyl (dialkyl) methoxy or 4-substituted-2-alkyl-1,3-dioxolane-2-yl bond group can be cleaved by H ₃ O ⁺ ; The 2-silylethoxy bond group may be cleaved by fluorine or an acid; 2- (X) -ethoxy, where X is a keto, ester amide, cyano, NO ₂ , sulfide, sulfoxide, sulfone bond group can be cleaved under alkaline conditions; The 2-, 3-, 4-, 5- or 6-substituted-benzyloxy bond group may be cleaved under acid or reducing conditions; The 2-butenyloxy linkage group can be cleaved by (Ph ₃ P) ₃ PhCl (H); The 3-, 4-, 5- or 6-substituted-2-bromophenoxy bond group may be cleaved by Li, Mg or BuLi; The methyl thiomethoxy bond group can be cleaved by Hg ²⁺ ; 2- (X) -ethyloxy (wherein X is a halogen) bond group may be cleaved by Zn or Mg; The 2-hydroxyethyloxy linkage group can be cleaved by oxidation (e.g., Pb (OAc) ₄ );
A preferred linker is a linker that is cleaved by acid or photolysis. Several acid-labile linkers that have been developed for solid phase peptide synthesis are useful for binding tack to MOI. Some of these linkers are described in a recent article by Lloyd-Wiliams et al. (Tetrahedron 49: 11065-1113, 1993). One useful form of linker is based on p-alkoxybenzyl alcohol and two 4-hydroxymethylphenoxyacetic acid and 4- (4-hydroxymethyl-3-methoxyphenoxy) butyric acid are commercially available [ Acvanced ChemTech (Louisville, KY). Both linkers can be attached to the tack via ester linkages to the benzyl alcohol and attached to the MOI containing amines via amide linkages to the carboxylic acid. The bonds bound by these molecules are released from the MOI with varying concentrations of trifluoroacetic acid. Carboxylic acid is released from the tack by the cleavage of these linkers. In a tack released by acid cleavage of the attached tack through an associated linker such as 2,4-dimethoxy-4 '- (carboxymethyloxy) -benzhydrylamine (manufacturer: Advanced ChemTech, FMOC-protected form) To induce the release of the carboxylamide.
Also, photo-labile linkers useful in these applications have been developed for the synthesis of solid phase peptides (see Lloyd-Williams review). These linkers are typically based on 2-nitrobenzyl esters or 2-nitrobenzyl amides. Two examples of photolabile linkers recently reported in the literature are: 4- (4- (1-Fmoc-amino) ethyl) -2-methoxy-5-nitrophenoxy) butanoic acid (Holmes and Jones, J (Fmoc-amino) -3- (2-nitrophenyl) propionic acid (Brown et al., Molecular Diversity 1: 4-12, 1995) . These two linkers are attached to the amino in the MOI via a carboxylic acid. Attachment of the tack to the linker is accomplished by forming an amine between the carboxylic acid in the tack and the amine in the linker. The cleavage of the photolabile linker is typically performed using UV light at a wavelength of 350 nm at an intensity and time known in the art. When the linker is cleaved, the amide of the free radicals is released first. Examples of photo-cleavable linkers include nitrophenyl glycine ester, exo- and endo-2-benzo norbornyl chloride and methanesulfonate and 3-amino-3 (2-nitrophenyl) propionic acid. Examples of cleavage by an enzyme include an esterase capable of cleaving an ester bond, a nuclease capable of cleaving a phosphodiester bond, a protease cleaving a peptide bond, and the like.
The preferred linker has an ortho-nitrobenzyl structure represented by the following formula (13)
In this formula,
One carbon atom at a, b, c, d or e position is replaced by -L ³ -X and L ¹ (preferably a direct bond) is present on the left side of N (R ¹ ) .
Such a linker can selectively cleave the bond between the carbon labeled " a " and N (R < ¹ >). R ¹ is typically selected from hydrogen and hydrocarbyl, although identification of R ¹ is typically not critical for the cleavage reaction. The present invention provides that in the above structure, -N (R ¹ ) - can be replaced by -O-. Also, in the above structure, one or more of the positions b, c, d or d may be optionally replaced by alkyl, alkoxy, fluorine, chlorohydroxy, carboxylate or amide, do.
A further preferred linker having the chemical handle L _h has the structure of Formula 14:
In this formula,
One or more of the b, c, d or e positions may optionally be replaced by alkyl, alkoxy, fluorine, chlorohydroxy, carboxylate or amide,
R < ^{1 >} is hydrogen or hydrocarbyl,
R ² is a group that protects or activates the carboxylic acid for coupling with OH or another moiety.
Fluorocarbons and hydrofluorocarbon groups are preferred groups for activating carboxylic acids for coupling with another moiety.
3. The molecule of interest (MOI)
Examples of MOIs include, but are not limited to, nucleic acids or nucleic acid analogs (e.g., PNA), fragments of nucleic acids (e.g., nucleic acid fragments), synthetic nucleic acids or fragments, oligonucleotides (e.g., DNA or RNA), proteins, Or antibody fragments, receptors, receptor ligands, members of ligand pairs, cytokines, hormones, oligosaccharides, synthetic organic molecules, drugs and combinations thereof.
A preferred MOI comprises nucleic acid fragments. A preferred nucleic acid fragment is a primer sequence complementary to a sequence present in a vector when the vector is used for sequencing the base. Preferably the nucleic acid fragment is attached directly or indirectly to the tack at a position other than the 3 ' end of the fragment; Most preferably at the 5 ' end of the fragment. Nucleic acid fragments may be purchased or may be prepared based on a genetic database (see, for example, Dib et al., Nature 380: 152-154, 1996 and CEPH Genotype Database, http://www.cephb.fr) (For example, Promega, Madison, Wis.).
As used herein, MOI includes derivatives of MOI containing functional groups useful in coupling MOI to TLL _h . For example, when a phosphodiester is bound to an alkylene amine, the nucleic acid fragment having a phosphodiester at the 5 ' end is an MOI. Such an MOI is described, for example, in U.S. Patent No. 4,762,779, which is incorporated herein by reference. The internally modified nucleic acid fragment is also an MOI. For example, internal modification of a nucleic acid fragment is when a base (e.g., adenine, guanine, cytosine, thymidine, uracil) is modified to add a reactive functional group. Such internally modified nucleic acid fragments are commercially available (manufacturer: Glen Research, Herndon, VA). Another example of internal modification of a nucleic acid fragment is where the phosphoramidate is used to synthesize a modified phosphodiester that is located between the sugar and phosphate groups of the nucleic acid fragment. The phosphoramidate contains a reactive group that allows the nucleic acid fragment containing such a phosphoramidate-derived moiety to be attached to another moiety, such as, for example, a TLH _h compound. These phosphoramidates are commercially available (manufacturer: Clonetech Laboratories, Inc., Palo Alto, Calif.).
4. Chemical Handle (L _h )
The chemical handle is a stable reactive aromatic moiety present as part of the first molecule, wherein the handle is capable of chemically reacting with a complementary chemical handle present as part of the second molecule to form a covalent bond between the two molecules. For example, the chemical handle may be a hydroxyl group, and the supplemental chemical handle may be a carboxylic acid group (or an activated derivative thereof, such as, for example, a hydrofluoroaryl ester) To form a covalent bond (especially an ester group) which bonds two molecules together.
The chemical handles can be used for a variety of covalent bond forming reactions suitable for attaching the tack to the linker and attaching the linker to the MOI. This reaction can be carried out in the presence of an alkylating agent (e.g., to form an ether, a thioether), an acylation (e.g., to form an ester, an amide, a carbamate, a urea, a thiourea), a phosphorylation (For example, to form sulfonates, sulfonamides), condensation (e.g., to form phosphates, phosphonates, phosphoramides, phosphonamides) To form a reactive intermediate such as nitrene or carbene by silylation, disulfide formation, and photolysis. Generally, the reaction to form a bond suitable for attaching the handle and tack to the linker is also suitable for attaching the linker to the MOI and vice versa. In some cases, the MOI may provide the necessary handle to attach the linker prior to modification or induction.
One type of bond that is particularly useful for attaching a linker to an MOI is a disulfide bond. The formation of this requires the presence of a thiol group (" handle ") in the linker and the presence of another thiol group at the MOI. A mild oxidation condition can couple two thiols together as a disulfide. In addition, the formation of the disulfide can be induced, for example, by using an excess of suitable disulfide exchanger such as pyridyl disulfide. Since the formation of the disulfide can be easily reversible, the disulfide, if desired, can be used as a cleavage bond to release the tack. This is typically accomplished under similar mild conditions using an excess of suitable disulfide exchanger such as dithiothreitol.
Amide bond formation is particularly interesting for attaching a tack (or tack with a linker) to an oligonucleotide. The primary aliphatic amine handle was synthesized using a phosphoramidite such as 6-monomethoxytrityl hexanoyl-N, N-diisopropylphosphoramidite (manufacturer: Glenn Research, Sterling, VA) Can be rapidly introduced onto the oligonucleotide. Amines found in natural nucleotides such as adenosine and guanosine are virtually unreactive when compared to primary amines introduced. This difference in reactivity forms the basic capability of selectively forming a linking group (e.g., urea, thiourea, sulfonamide) associated with the introduced primary amine rather than amide and nucleotide amines.
As listed in the Molecular Probes catalog (Molecular Probes catalog, Eugene, OR), a partial list of amine reactive functional groups includes active carboxyl esters, isocyanates, isothiocyanates, sulfonyl halides, and dichlorotriazenes. The active ester is an excellent preparation for the formation of amines because the amide product formed is very stable. In addition, these agents retain good reactivity with aliphatic amines and possess low reactivity with the nucleotide amines of the oligonucleotides. Examples of active esters include N-hydroxymaleimide esters, pentafluorophenyl esters, tetrafluorophenyl esters and p-nitrophenyl esters. Active esters are useful because they can be prepared from virtually any molecule containing a carboxylic acid. Methods for preparing active esters are described in Bodansky, Principles of Peptide Chemistry (2d ed.), Springer Verlag, London, 1993.
5. Attachment of Linker
Typically, a single form of linker is used to link a particular set or group of taps to a particular set or group of MOIs. In a preferred embodiment of the present invention, a single homogeneous process can be performed to form all the various T-L-MOI structures. This is particularly advantageous, especially when the set of T-L-MOI structures is large, since this allows for the fabrication of sets using methods of combinatorial chemistry or other parallel processing techniques. In a similar manner, using a single type of linker, a single uniform process can be used to cut all the various T-L-MOI structures. It is also advantageous for a large set of T-L-MOI structures because they can be processed in parallel, repeatedly and / or in an automated manner.
However, there are other embodiments of the invention in which two or more linkers are used to link different subsets of a tack to a subset of the corresponding MOI. In this case, using selective cutting conditions, each linker can be cut independently without cutting the linkers present in the subset of other MOIs.
Many reactions that form covalent bonds are suitable for attaching the tack to the linker and the linker to the MOI. This reaction can be carried out in the presence of an alkylating agent (e.g., to form an ether, a thioether), an acylation (e.g., to form an ester, an amide, a carbamate, a urea, a thiourea), a phosphorylation (For example, to form sulfonates, sulfonamides), condensation (e.g., to form phosphates, phosphonates, phosphoramides, phosphonamides) To form a reactive intermediate such as nitrene or carbene by silylation, disulfide formation, and photolysis. Generally, the reaction to form a bond suitable for attaching the handle and tack to the linker is also suitable for attaching the linker to the MOI and vice versa. In some cases, the MOI may provide the necessary handle to attach the linker prior to modification or induction.
One type of bond that is particularly useful for attaching a linker to an MOI is a disulfide bond. The formation of this requires the presence of a thiol group (" handle ") in the linker and the presence of another thiol group at the MOI. A mild oxidation condition can couple two thiols together as a disulfide. In addition, the formation of the disulfide can be induced, for example, by using an excess of suitable disulfide exchanger such as pyridyl disulfide. Since the formation of the disulfide can be easily reversible, the disulfide, if desired, can be used as a cleavage bond to release the tack. This is typically accomplished under similar mild conditions using an excess of suitable disulfide exchanger such as dithiothreitol.
The formation of amide bonds is of particular interest for tacking and binding to nucleotides. The primary aliphatic amine handle was synthesized using a phosphoramidite such as 6-monomethoxytrityl hexanoyl-N, N-diisopropylphosphoramidite (manufacturer: Glenn Research, Sterling, VA) Can be rapidly introduced onto the oligonucleotide. Amines found in natural nucleotides such as adenosine and guanosine are virtually unreactive when compared to primary amines introduced. This difference in reactivity forms the basic capability of selectively forming a linking group (e.g., urea, thiourea, sulfonamide) associated with the introduced primary amine rather than amide and nucleotide amines.
As listed in the Molecular Probes catalog (Molecular Probes catalog, Eugene, OR), a partial list of amine reactive functional groups includes active carboxyl esters, isocyanates, isothiocyanates, sulfonyl halides, and dichlorotriazenes. The active ester is an excellent preparation for the formation of amines because the amide product formed is very stable. In addition, these agents retain good reactivity with aliphatic amines and possess low reactivity with the nucleotide amines of the oligonucleotides. Examples of active esters include N-hydroxymaleimide esters, pentafluorophenyl esters, tetrafluorophenyl esters and p-nitrophenyl esters. Active esters are useful because they can be prepared from virtually any molecule containing a carboxylic acid. Methods for preparing active esters are described in Bodansky, Principles of Peptide Chemistry (2d ed.), Springer Verlag, London, 1993.
There are a number of commercially available crosslinkers, which can act as linkers (Pierce Cross-linkers, Pierce Chemical Co., Rockford, Ill.). Among these are homogeneous bifunctional amine reactive crosslinking agents such as, for example, homobifunctional imidoesters and N-hydroxysuccinimidyl (NHS). Also included are heterobifunctional crosslinking agents having two or more different reactive groups that allow for sequential reactions. The imides readily react with amines at alkaline pH. The NHS-ester provides a stable product when reacted with a primary or secondary amine. The maleimide, alkyl and aryl halides, alpha-haloacyl and pyridyl disulfides are thiol reactive. Maleimide can be specific for the thiol (sulfhydryl) group in the pH range of 6.5 to 7.5 and amine reactive at alkaline pH. The thioether linkages are stable under physiological conditions. The alpha-haloacetyl cross-linker contains iodoacetyl groups and is reactive to sulfhydryls. Imidazole can react with the iodoacetyl moiety, but the reaction is very slow. The pyridyl disulfide reacts with the thiol group to form a disulfide bond. The carbodiimide couples the carboxyl to the hydrazide primary amine causing the formation of an acyl-hydrazine bond. Aryl azide is a chemically inert photoinitiator until exposed to UV or visible light. When such a compound is photodecomposed at 250 to 460 nm, reactive arylene nitrile is formed. Reactive arylnitrenes are relatively nonspecific. Glyoxal is reactive to the guanidinyl moiety of arginine.
In one exemplary embodiment of the invention, the tack is first bound to a linker, and then the tack and linker combination is combined with the MOI to form a T-L-MOI structure. In addition, the same structure is formed by first binding the linker to the MOI, and then combining the linker with the MOI combination. This is an example in which the MOI is a DNA primer or oligonucleotide. In this case, the tack is typically first coupled to a DNA primer or oligonucleotide that is then coupled to a linker, T-L, which is used, for example, in a sequencing reaction.
One useful form (e. G., Oligonucleotide or DNA sequencing primer) that is attached to the MOI in a tacky fashion is through a chemically labile linker. A preferred design for the linker allows the linker to be cleaved when exposed to, for example, volatile organic acids such as trifluoroacetic acid (TFA). In particular, TFA is compatible with most MS ionization methods, including electrospray.
As described in detail below, the present invention provides a method for determining the shape of a gene. Compositions useful in the method for determining the morphology of a gene include a number of compounds of formula 15:
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride decomposition, and any element selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows the residue containing T ^ms to be cleaved from the residue of the compound, wherein the residue containing T ^ms is a compound wherein the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids A functional group that supports a single ionized charge state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI. In a composition, two or more compounds have the same T ^ms , but the MOI group of such molecules has a non-identical nucleotide length.
Another composition useful in the method of determining the genomic shape comprises a plurality of compounds of formula 15:
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any element selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows the residue containing T ^ms to be cleaved from the residue of the compound, wherein the residue containing T ^ms is a compound wherein the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids A functional group that supports a single ionized charge state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI. In the composition, two or more compounds have the same T ^ms , but these compounds have an unequal elution time by chromatography.
Another composition that can be used in the method of determining the genetic shape comprises a plurality of compounds of formula 15:
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any element selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows the residue containing T ^ms to be cleaved from the residue of the compound, wherein the residue containing T ^ms is a compound wherein the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids A functional group that supports a single ionized charge state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI. In the composition, the two compounds having the same length of the MOI nucleotide do not have the same T ^ms .
In the above composition, the majority preferably exceeds 2, and more preferably exceeds 4. Further, the nucleic acid fragment in the MOI has a sequence complementary to a portion of the vector, wherein the fragment can stimulate the synthesis of the polynucleotide. Preferably, the T ^ms group in the plurality of members is at least 2 amu different and may be at least 4 amu different.
The present invention also provides a composition wherein each compound comprises a plurality of sets of compounds of Formula 15:
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any element selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows the residue containing T ^ms to be cleaved from the residue of the compound, wherein the residue containing T ^ms is a compound wherein the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids A functional group that supports a single ionized charge state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI. In the composition, the members in the first set of compounds have the same T ^ms group but different members of the nucleotide in the MOI, with the same MOI group and there are at least 10 members in the first set, The T ^ms group is more than 2 amu different. The majority is preferably 3 or more, and more preferably 5 or more.
The present invention also provides a composition wherein each compound comprises a plurality of sets of compounds of Formula 15:
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any element selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows the residue containing T ^ms to be cleaved from the residue of the compound, wherein the residue containing T ^ms is a compound wherein the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids A functional group that supports a single ionized charge state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI. In the composition, the compounds in the set have the same elution time but the ^Tms group is not the same.
The present invention also provides a kit for determining the shape of a gene comprising at least one primer pair of primer pairs of the following formula (15), wherein each primer pair is related to a different position:
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any element selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows the residue containing T ^ms to be cleaved from the residue of the compound, wherein the residue containing T ^ms is a compound wherein the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids A functional group that supports a single ionized charge state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI. In the kit, the majority is preferably 3 or more, more preferably 5 or more.
As indicated above, the present invention provides compositions and methods for determining the sequence of nucleic acid molecules. Briefly, this method generally comprises the steps of (a) forming a tacked nucleic acid in a nucleic acid molecule at a second terminus that replenishes a nucleic acid molecule selected from the first terminus (e. G., A tacked fragment) Correlates with a particular nucleic acid fragment and can be detected by any of a variety of methods;
(b) separating said tagged fragment by the length of the sequence;
(c) cutting the tack from the tacked piece;
(d) determining the identity of the nucleic acid molecule, including detecting the tag, and determining the sequence of the nucleic acid molecule therefrom. Each of these aspects is described in more detail below.
B. Diagnostic Methods
1. Introduction
As noted above, the present invention provides a wide variety of methods, wherein the tack and / or linker described above may be used in place of conventional markers to enhance the specificity, sensitivity, or number of samples that can be analyzed simultaneously within a given method (E. G., By radioactivity or by enzymes). Representative examples of such methods that can be enhanced include, for example, RNA amplification (see Lizardi et al., Bio / Thchnology 6: 1197-1202, 1988; Kramer et al., Nature 339: 401-402, 1989; Lomeli et al., Clinical Chem. 35 (9): 1826-1831, 1989; U.S.A. Patent No. 4,786,600), and DNA amplification using LCR or polymerase chain reaction (" PCR "). Patent Nos. 4,683,195, 4,683,202 and 4,800,159.
In one embodiment of the present invention, there is provided a method of producing a nucleic acid molecule comprising: (a) generating a nucleic acid molecule selected from one or more selected target nucleic acid molecules, wherein the tag is correlated with a particular nucleic acid fragment and detected by non-fluorescence spectroscopy or potentiometry ≪ / RTI > (b) separating the set molecules by size; (c) cutting the tack from the molecule with the tack set; (d) determining the identity of the nucleic acid molecule or fragment, or detecting the presence of the selected nucleic acid molecule or fragment, including detecting the tag by non-fluorescence spectrometry or potentiometry and then determining the identity of the nucleic acid molecule therefrom; . ≪ / RTI > .
In a related aspect of the present invention, there is provided a method for detecting a nucleic acid molecule comprising: (a) combining a nucleic acid probe with a target nucleic acid molecule, wherein the nucleic acid probe is tiled for a sufficient time and under conditions permitting hybridization of the nucleic acid probe tacked to the target nucleic acid sequence selected complementarily; At this time, the tacked nucleic acid probe can be detected by non-fluorescence spectroscopy or potentiometry; (b) varying the size of the hybridized and tapped probe, the size of the unhybridized probe or target molecule, or the size of the probe: target hybrid; (c) separating the tacked probe by size; (d) cutting the tack from the tacked probe; (e) detecting a tack by non-fluorescence spectroscopy or potentiometric methods, and then detecting a nucleic acid molecule selected therefrom. These and other related techniques are described in further detail below.
2. PCR
PCR can amplify the desired DNA sequence of any organism (virus, bacterium, plant or person) to millions of times over time. PCR is particularly useful because the reactions are highly specific, particularly automated, and can amplify very small amounts of sample. For these reasons, PCR plays an important role in clinical medicine, hereditary disease diagnosis, forensic science and environmental biology.
Briefly, PCR is a process based on a particular polymerase, which is a process that replaces a given DNA strand flanking a target sequence in a mixture containing four DNA bases and two DNA fragments (primers, each about 20 base lengths) Can be synthesized. The mixture is heated to isolate the strands of the double-stranded DNA containing the target sequence and then cooled to (1) discover the primers and bind their complementary sequences to the separated strands, and (2) It allows to extend. Repeated heating and cooling cycles exponentially expand DNA because they are separated into two templates for the synthesis of each new diblock chain. After about 1 hour, 20 PCR cycles can amplify the target up to a million fold.
Within one embodiment of the invention there is provided a method for determining the identity of a nucleic acid molecule, for example using a PCR technique in a biological sample, or for detecting a selected nucleic acid molecule. Briefly, the method comprises forming a series of tactile nucleic acid fragments or molecules during PCR and separating the fragments produced by size. The size separation step may be accomplished using any of the techniques described herein, including, for example, gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC. The tack is then cut from the separated fragments and detected by respective detection techniques. Examples of such techniques are described herein and include, for example, mass spectrometry, infrared spectroscopy, constant potential current or UV spectrometry.
3. RNA fingerprinting and deviation display
If the template is RNA, the first step in fingerprinting is reverse transcription. Although Liang and Pardee (Science 257: 967, 1992) used primers for reverse transcription based on oligo (dT), but at the 5 'end (eg oligo 5' - (dT ₁₁ ) CA-3 (RA) portion of the 12 polyadenylated RNAs selectively accessible from the 12 polyadenylated RNAs and a 5 ' -upG-poly < RTI ID = 0.0 > After reverse transcription and denaturation, the primers are subjected to independent stimulation on the first strand of the cDNA to be produced. PCR is now best matched to the primer and the mRNA and polyadenylated heterologous RNA Can be used to form a fingerprinting of the product derived from the 3 ' end. This protocol is termed " differential display ".
Also, proprietary primers can be used in the first step of reverse transcription in which 6-8 bases select an internal region for RNA that matches the 3 ' end of the primer. This is done by independent stimulation of the first strand of cDNA prepared using the same or different proprietary primers followed by PCR. This particular protocol sample is present in any of the RNAs containing an open reading frame (see Welsh et al., Nuc. Acids. Res. 20: 4965, 1992). It can also be used in non-polyacetylated RNA, like many bacterial RNAs. Such a modification of the RNA fingerprinting by the independently stimulated PCR is referred to as RAP-PCR.
When the independently stimulated PCR fingerprinting of RNA is performed on samples derived from cells, tissues or other biological material that have been experimentally treated or developed differently, differences in expression of the gene between the samples can be observed . In each case, the same number of effective PCR replication phenomena occurs and any difference in initial concentration of the cDNA product is maintained as a ratio of intensity in the final fingerprinting. There is no significant relationship between the intensities of the bands in a single lane in the gel, which is a function of the conjugate and the remainder. However, the ratio between lanes allows to detect RNA expressed differently for each sampled RNA. The ratio of starting material between samples is maintained even if the number of cycles is sufficient to permit PCR reactions to saturate. This is almost completely controlled by the products that form the majority fingerprinting, the number of duplications required to reach saturation. In this regard, PCR fingerprinting differs from conventional PCR of a single product in that the ratio of the starting material between the samples is maintained without sampling the product as exponential amplification.
Within one embodiment of the method according to the present invention, there is provided a method for determining the identity of a nucleic acid molecule, for example using RNA fingerprinting in a biological sample, or for detecting a nucleic acid molecule selected. Briefly, this method comprises forming a series of nucleic acid fragments to which the tag is attached. Fragments produced by PCR or by analogous amplification schemes are sequenced by size. The size separation step may be accomplished using any of the techniques described herein, including, for example, gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC. The tack is then cut from the separated fragments and detected by respective detection techniques. Examples of such techniques include, for example, mass spectrometry, infrared spectroscopy, constant potential current or UV spectrometry. The amount of each of the given provided nucleic acid fragments is not critical, but the size of the band provides information when referenced to the control sample.
4. Fluorescence PCR Polymerase Chain Polymorphism (PCR-SSCP)
In addition to the RFLP method, a number of methods can be used to analyze base-substituted polymorphs. Orita et al. Provide a method for analyzing these polymorphisms based on the confirmation bias in the denatured DNA. A relatively small DNA fragment is produced which is denatured by simply using restriction enzyme specific PCR and then degraded by electrophoresis on unmodified polyacrylamide gel. Confirmation deviations in a single-stranded DNA fragment prepared from base substitution are detected by the electrophoretic mobility axis. The intramolecular base pairs form a high degree of sequence-specific and discriminatory Japanese chain identity in the electrophoretic mobility. However, in different studies using conventional SSCP, the detection rate ranges from 35% to almost 100% with the highest detection rate, which usually requires several different conditions. In principle, this method can be used to analyze polymorphisms based on short insertions or deletions. This method is one of the most powerful tools for detecting point mutations and deletions in DNA (see SSCP-PCR Dean et al., Cell 61: 863, 1990).
Within one embodiment of the method according to the invention, there is provided a method for determining the identity of a nucleic acid molecule, for example using a PCR-SSP technique in a biological sample, or for detecting a nucleic acid molecule selected. Briefly, this method comprises forming a series of nucleic acid fragments to which the tag is attached. The fragments formed by PCR are then sequentially separated by size. Preferably the size separation step is unmodified and the nucleic acid fragment is denatured prior to the separation method. The size separation step may be accomplished using, for example, gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC techniques. The tack is then cleaved from the separated fragments and the tack is then detected by a respective detection technique (e.g., mass spectrometry, infrared spectroscopy, constant potential current method or UV spectrometry).
5. Dideoxy fingerprinting (ddF)
Another method described in the literature (ddF, Sarkar et al., Genomics 13: 441, 1992) detects 100% single-base changes in human IX gene factors when tested in reverse and forward mode. When genomic DNA is analyzed from patients using hemophilia B, a total of 84 of 84 different sequence changes are detected.
Briefly, in applying a tag for genotyping or other purposes, one method that can be used is dideoxy-fingerprinting. This method uses a dideoxy terminator in Sanger's sequencing reaction. The principle of this method is as follows: The target nucleic acid to be sequenced is placed in a reaction carrying a dideoxy-terminal terminator which replenishes a known base mutated in the target nucleic acid. For example, if an A → G change occurs by mutation, the reaction may be performed in a C-dideoxy-terminal terminator reaction. PCR primers are used to position and amplify the target sequence of interest. If the fictitious target sequence contains an A to G variation, the size of the multiple sequences will vary because they incorporate the dideoxy-terminal terminator into the amplified sequence. In the particular application of such a tack, in the case of a mutation, a fragment can be formed that has a predictable size. The tag can attach to the 5'-end of the PCR primer and can provide a "map" to the morphology of the sample and the dideoxy-terminal terminator form. PCR amplification reactions can be performed to produce fragments that can be separated by size, e. G., By HPLC or PAGE. At the end of the separation process, the DNA fragment is collected in a temporary reference frame, and the appearance or absence of the mutation after truncation of the tack is determined by chain length due to the incorporation of the provided dideoxy-terminal terminator by the early strand terminator .
Importantly, it has been found that ddF causes the loss or gain of the axis in the dynamics of the dideoxy-terminal segment or of one or more terminal segments or products. Thus, in this method, the axis of mobilization of one fragment is examined at a high background of other molecular weight fragments. There is an advantage of knowing in advance the length of the fragment associated with the provided mutation.
Within one embodiment of the method according to the invention, there is provided a method for determining the identity of a nucleic acid molecule, for example, using a ddF technique in a biological sample, or for detecting a nucleic acid molecule selected. Briefly, such methods include separating by size followed by the step of forming a series of nucleic acid fragments. Preferably the size separation step is unmodified and the nucleic acid fragment is denatured prior to the separation method. The size separation step may be accomplished using, for example, gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC techniques. The tack is then cleaved from the separated fragments and the tack is then detected by a respective detection technique (e.g., mass spectrometry, infrared spectroscopy, constant potential current method or UV spectrometry).
6. Restriction maps and RFLP
Restriction endonuclease recognizes short DNA sequences and cleaves DNA molecules at specific sites. Some restriction enzymes (rare cleavage agents) very often cut DNA to form a small number of very large fragments (thousands to millions of bp). Most enzymes cleave DNA more frequently to form the majority of small fragments (bp from one hundred to one thousand). On average, a restriction enzyme with a 4-base recognition site forms a length of 256 bases, a 6-base recognition site forms 4000 bases in length, and an 8-base recognition site forms 64,000 bases in length. Because hundreds of different restriction enzymes are characterized, DNA can be cleaved into a number of different small fragments.
A wide variety of techniques have been developed for analyzing DNA polymorphisms. The most widely used method, the restriction fragment length polymorphism (RFPL) method combines restriction enzyme digestion, gel electrophoresis, contamination of membranes and hybridization to a cloned DNA probe. The polymorphism is detected as a change in the length of the labeled fragment on the contaminant. When the change in sequence is attributed to the restriction enzyme site using the RFLP method, it is possible to analyze the base substitution or to analyze the minisatellites / VNTR by selecting a restriction enzyme that cleaves the outside of the repeat unit. Agarose gels generally do not allow the necessary degradation to distinguish small minor / VNTR alleles that vary by a single repeat unit, but a number of small minor / VNTRs are of such value that they can obtain highly beneficial markers .
Within one embodiment of the method according to the invention there is provided a method for determining the identity of a nucleic acid molecule or for detecting a selected nucleic acid molecule using, for example, restriction map formation or RFLP techniques in a biological sample. Briefly, this method comprises forming a series of tactile nucleic acid fragments in which the fragment to be formed is degraded by a restriction enzyme. The tagged fragment is formed by performing the hybridization step of the probe selected using the degraded target nucleic acid. The hybridization step may be performed prior to or after the restriction nuclease degradation. The degraded nucleic acid fragments produced are separated by size. The size separation step may be accomplished using, for example, gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC techniques. The tack is then cleaved from the separated fragments and the tack is then detected by a respective detection technique (e.g., mass spectrometry, infrared spectroscopy, constant potential current method or UV spectrometry).
7. DNA fingerprinting
DNA fingerprinting involves the display of a set of DNA fragments from a particular DNA sample. Various DNA fingerprinting techniques currently available utilize PCR to form fragments (see Jeffries et al., Nature 314: 67, 1985; Welsh and McClelland, Nuc. Acids. Res. 19: 861, 1991 ). The fingerprinting techniques to be used are chosen, for example, depending on the application, such as DNA typing, DNA marker maps and organisms under irradiation conditions such as, for example, prokaryotes, plants, animals and humans. Many fingerprinting methods that meet these requirements have been developed over the past several years, including randomly amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF) and optionally stimulated PCR (AP-PCR) come. These methods are all based on the amplification of irregular genomic DNA fragments by randomly selected PCR primers. The DNA fragment pattern can be formed in any DNA without knowledge of the sequence of the dictionary. The pattern to be formed depends on the sequence of the PCR primer and the nature of the template DNA. PCR is performed at annealing temperature to anneal the primers to overlap positions in the DNA. DNA fragments are formed when the primer binding site is within a distance that allows amplification. In principle, a single primer is sufficient to form a pattern of bands.
A novel technique for DNA fingerprinting has been described as AFLP (Voc et al., Nuc. Acids Res. 23: 4407, 1995). The AFLP technique is based on the detection of genomic restriction fragments by PCR amplification and can be used for DNA of any source or complex. Briefly, fingerprints are formed without prior sequence knowledge using a limited set of gene primers. Multiple fragments detected in a single reaction may be " turns " by selection of a particular primer set. AFPL technology is robust and reliable because stringent reaction conditions are used for primer annealing: the reliability of RFLP technology is combined with PCR technology.
In one embodiment of the method according to the present invention, for example, a method for determining the identity of a nucleic acid molecule, or detecting a nucleic acid molecule selected, using a DNA fingerprinting technique in a biological sample is provided. Briefly, such methods include separating by size followed by the step of forming a series of nucleic acid fragments. Preferably, the size separation step can be accomplished using, for example, gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or preferably HPLC techniques. The tack is then cleaved from the separated fragments and the tack is then detected by a respective detection technique (e.g., mass spectrometry, infrared spectroscopy, constant potential current method or UV spectrometry).
8. Application of truncation tack to genotype and polymorphic detection
a. Introduction
The majority are based on single base substitutions or multiple iterations, although a small number of known human DNA polymorphisms are based on the insertion, deletion or other rearrangement of sequences that do not repeat. The base substitution is very sufficient in the human genome and occurs once every 200 to 500 bp on average. The change in length in a block of series repeats is common in the genome along with dozens of thousands of scattered polymorphic sites (named position). The repeat length for the serially repeating polymorphism ranges from 1 bp in the (dA) _n (dT) _n sequence to 170 bp or more in the a-side DNA. The serially repeating polymorphism consists of a small minor number / variable number of tandem repeats (VNTR = variable number of tandem repeats) with 10 typical repeats in base pairs and several thousand repeating units and a repeat length of 6 bp, And a microsphere whose total length is about 70 bp. Most of the microsatellite forms defined for the data are based on (dC-dA) _n or (dG-dT) _n dinucleotide repeat sequences. Polymorphic analysis of the microsatellite involves polymerase chain reaction (PCR) of small fragments of DNA containing the repeat block followed by amplification of the amplified DNA by electrophoresis of the polyacrylamide gel by denaturing the polyacrylamide gel. The PCR primer replenishes a unique sequence that flank repeating blocks. Polyacrylamide gels that are not agarose gels are typically used for microsaturation because the alleles differ only in size by a single repetition.
Thus, within one aspect of the invention, there is provided a method of detecting a nucleic acid molecule comprising the steps of: (a) forming a nucleic acid molecule that is tacked from a selected target molecule, wherein the tack is correlated with a particular nucleic acid fragment, Can be; (b) separating the set molecule by the length of the sequence; (c) cutting the tack from the molecule with the tack set; (d) detecting a tack by non-fluorescence spectroscopy or potentiometric methods, and then determining the genotype of the organism from which the gene form of the selected organism is determined.
In yet another embodiment, a method is provided that comprises: (a) combining a nucleic acid molecule selected for a sufficient time and under conditions that allow for hybridization of a molecule set tacked to the target molecule with the selected target molecule, And can be detected by non-fluorescence spectroscopy or by potentiometry; (b) separating the set molecule by the length of the sequence; (c) cutting the tack from the molecule with the tack set; (d) detecting a tack by non-fluorescence spectroscopy or potentiometric methods, and then determining the genotype of the organism from which the gene form of the selected organism is determined.
b. Application of truncable tack to gene shape determination
The PCR method for identifying restriction fragment length polymorphisms (REPLs) combines the detection of gel electrophoresis and tacking associated with specific PCR primers. In general, one PCR primer can have one specific tag. Thus, a tag can represent one set of PCR primers and the length of a predetermined DNA fragment. The polymorphism is detected as a change in the length of the labeled fragment in the gel. Polyacrylamide gel electrophoresis allows for the necessary degradation to distinguish small minority / VNTR alleles that typically vary by a single repeating unit. The analysis of microsaturated morphology was performed by polymerase chain reaction (PCR) of a small fragment of DNA containing a repetitive block followed by denaturation of the polyacrylamide gel, followed by electrophoresis of amplified DNA or separation of DNA fragments by HPLC Lt; / RTI > The amplified DNA may be labeled using a primer having a tag cleavable at the 5 ' end of the primer. The primer is incorporated into the newly synthesized strand by chain extension. The PCR primer replenishes a unique sequence that flank repeating blocks. The small ancillary / VNTR polymorphism can be amplified as much as using the microsatellites described above.
DNA Sequence Describes the fundamental basis for understanding the genome structure of humans on many forms of polymorphism (see, for example, Botstein et al., Am. J. Human Genetics 32: p314, 1980; Donis-Keller, 319, 1987; Weissenbach et al., Nature 359: 794). The construction of the combinatorial map of the extended structure is facilitated by using such DNA polymorphisms and provides a practical means for localizing disease genes by binding. The dinucleotide marker of the microsatellite proves to be a very powerful instrument in identifying genes in humans that contain mutations and, in some cases, cause disease. The genomic dinucleotide repeats are polymorphic (see Weber, 1990, Genomic Analysis, Vol 1, pp 159-181, Cold Spring Laboratory Press, Cold Spring Harbor, NY; Weber and Wong, 1993, Hum. . p1123) may have fewer than 24 alleles. The microsatellite dinucleotide repeats can be amplified by PCR using primers that complement the unique region surrounding the dinucleotide repeat. After amplification, some amplified positions are compounded (compounded) prior to the size separation step. The process of applying the amplified microsatellite fragments to the size separation step and identifying size and alleles is known as determining gene shape. High complexity chromosome specificity markers have been reported to perform a whole genome scan for binding analysis (Davies et al., 1994, Nature, 371, p130).
Tacs can be used effectively to determine the genotype using microsatellite. Briefly, PCR primers are constructed to retain tack and used in carefully selected PC reactions to amplify the di-, tri- or tetra-nucleotide repeats. The amplification products are then separated by size, such as by HPLC or PAGE. The DNA fragments are collected in a transient manner and the tack is collected from each DNA fragment and the length deduced from comparison to the internal standard in the size separation step. Identification of the identity of an allele is made from a reference to the size of the amplified product.
Multiple samples can be combined in a single separation step using a truncable tack for the determination of the gene type. There are two general ways to do this. The first common approach for high-throughput screening is to detect a single polymorphism with a large group of individual substances. In this manner, a single or set of PCR primers are used and each amplification is done using one DNA sample form per reaction. The number of samples that can be blended in the separation step is proportional to the number of cleavable tacks that can be formed on the detection technique (for example, 400 to 600 for mass spectrometry tack). Hence, one to several polymorphs can be identified in large groups of individual substances at the same time. The second approach is to use multiple sets of PCR primers (for example, determination of individual gene types) that can identify multiple polymorphisms on a single DNA sample. In this manner, PCR primers are combined in a single amplification reaction to form PCR products of different lengths. Each primer pair or set is encoded by a specific truncable tag that can encode each PCR fragment with a specific tag. The reaction is carried out in a single separation step (see below). The number of samples that can be blended in the separation step is proportional to the number of cleavable tacks that can be formed per detection technique (e.g., 400-600 for a mass spectrometry tack).
c. Detection and application of mutations by enzymes
In this particular application and method, mismatches in the heteroduplex are detected by cleavage of the mismatched base pairs by enzymes in the provided nucleic acid duplication. The DNA sequence to be tested for the presence of the mutation is amplified by PCR using a specific set of primers and the amplified product is mixed with the denatured and denatured reference fragment and hybridized to form a heteroduplex. The heteroduplex then recognizes the duplicates in the presence of mismatches and treats with the cleaved enzyme. These enzymes are nuclease S1, malignant nuclease, " resolebase ", T4 endonuclease IV, and the like. Essentially any enzyme that recognizes mismatches in vitro can be used and mismatches formed can be cleaved. Treatment using appropriate enzymes, DNA duplicates are separated by size, for example by HPLC or PAGE. Temporarily collect DNA fragments. Cut and detect tack. The presence of a mutation is detected by the axis in the movement of the fragment relative to the reference fragment in an uncorrected form.
d. Application of tack for oligonucleotide linkage assay (OLA)
Oligonucleotide linkage assays as described in the original literature (Landegren et al., Science 241: 487, 1988) are useful techniques for identification of (known) sequences in very large and complex genomes. The principle of the OLA reaction is based on the ability of the ligase to covalently couple two diagnostic oligonucleotides as they hybridize adjacent to one another on a given DNA target. The sequences at the probe junctions are not fully base paired and the probe can not bind by ligase. The ability of the thermostable ligase to differentiate the base-pair deviation of the dislocation signal when located at the 3'end of the " upstream " probe provides an opportunity for single base-pair resolution (Barony, PNAS USA 88: 189, 1991). In the application of tack, the tack may be attached to a probe connected to the amplified product. After completion of the OLR, the fragments were separated based on size, and the tack was cut and then detected by mass spectrometry.
e. Sequence specificity amplification
One or other alleles may be selectively amplified using a PCR primer having a 3 ' end complementary to a mutant oligonucleotide sequence or a normal oligonucleotide sequence (see Newton et al., Nuc. Acids Res., , Soma et al., 1989, Mayo Clin. Proc., 64 (1984), pp. 17, p2503; et al., 1989, Genomics, 5, p535; Okayama et al., 1989, J. Lab. , 1361; Wu et al., PNAS USA, 86, p2757). Typically, PCR products are visualized after amplification by PAGE, but the principle of sequence-specific amplification is applicable to solid-phase formats.
f. A strong application of the tack on the test based on several amplifications
Determining the genotype of a virus: The strongest application of a tag is to determine the genotype of the virus or identity of the virus by hybridization using a tacked probe. For example, the F + RNA E. coli phage may be a useful substance as an indicator for viral contamination of the small intestine. Determination of the genotype by the nucleic acid hybridization method is a reliable, rapid, simple and inexpensive serotype crystal (Kafatos et al., Nucleic Acids Res. 7: 1541, 1979). The amplification technique and the nucleic acid hybrid technique are shown in FIG. (See, for example, E. coli Feng, Mol. Cell Probes 7: 151, 1993), rotavirus (Sethabutr et al., J. Med Virol. 37: 192,1992), hepatitis C virus ; Various microorganisms including the herpes simplex virus (Matsumoto et al., J. Virol. Methods 40: 119, 1992), Stuyver et al., J. Gen. Virol. Have been successfully used for classification.
Prognostic Application of Mutation Assay in Cancer: Changes in genes in various experimental mammalian and human tumors have been described and represent a morphological basis for the sequence of morphological changes observed in carcinogenesis (see Vogelstein et < RTI ID = 0.0 > al., NEJM 319: 525, 1988). Recent advances in molecular biology techniques have led to the loss of alleles in mutations in certain oncogenes (eg, c-myc, c-jun and rat families) as well as specific chromosomes or mutations in tumor suppressor genes The nature of research. Previous work (Finkelstein et al., Arch Surg. 128: 526, 1993) confirms the correlation between the specific forms of point mutations in K-rat oncogenes and the diagnostic stages of colorectal carcinoma. The results suggest that tumor aggregation, including the pattern and distribution of metastases, can provide important information by mutation analysis. The prognostic value of TP53 and K-ras-2 mutation analysis in the third stage of colon has recently been shown (Pricolo et al., Am. J. Surg. 171: 41, 1996). Therefore, it is clear that the determination of the genotype of tumor and prospective carcinoma cells and the detection of a specific mutation will become more important in the treatment of cancer in humans.
C. Isolation of nucleic acid fragments
Samples that require analysis are often a mixture of multiple components in a composite matrix. For samples containing unknown components, the components should be separated from each other so that each individual component can be identified and stuck by other analytical methods. The separation characteristics of the components in the mixture are constant under certain conditions and, once determined, they can be used to identify and measure each component. This process is typical for chromatographic and electrophoretic separation.
1. High Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography (HPLC) is a chromatographic separation technique for separating compounds that are soluble in solution. The HPLC apparatus consists of a reservoir, a pump, an injector, a separation column and a detector of the fluid phase. The compound is separated by injecting an aliquot of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to their deviation in their distribution behavior between the flowing liquid phase and the stagnation phase.
Recently IP-RO-HPLC on non-porous PS / DVB particles with chemically bonded alkyl chains has been shown to provide a similar degree of solubility and a rapid alternative to capillary electrophoresis in the analysis of diblock chains ( (Huber et al., Biotechniques 16: 898, 1993). In contrast to ion-exchange chromatography, which does not always retain double-stranded DNA as a function of chain length (since the AT base pair interacts more strongly than the GC base pair with the positively-charged stoichiometry), IP-RO- - Dependent separation is possible.
A method in which phosphodiester oligonucleotides can be successfully separated by high performance liquid chromatography on non-porous 2.3 .mu.M poly (styrene-divinylbenzene) particles alkylated using 100 mM triethylammonium acetate as an ion-pair preparation (Oefner et al., Anal. Biochem. 223: 39, 1994). The techniques described allow for the isolation of PCR products that differ only in base pairs from 4 to 8 nucleotides in length within the size range of 50 to 200 nucleotides.
2. Electrophoresis
Electrophoresis is a separation technique based on the mobility of ions (or DNA, as described herein) in an electric field. The negatively charged DNA moves towards the positive electrode and the positively-charged ions move toward the negative. For safety reasons, one electrode is typically on the ground and the other is biased positively or negatively. Charged materials may have different rates of motion depending on their overall charge, size and shape and may thus be separated. The electrode arrangement comprises a high voltage power supply, electrodes, buffers and supports for buffers such as polyacrylamide gel or capillary tubes. Open capillary tubes are used for many types of samples and other gel supports are typically used for biological samples such as protein mixtures or DNA fragments.
3. Capillary electrophoresis (CE)
Capillary electrophoresis (CE) in a variety of manipulations (glass solution, isotaxopoiesis, isoelectric focusing, polyacrylamide gel, micellar electrophoresis "chromatography") is a method for rapid high resolution separation of complex mixtures of very small volumes . With the combination of MS's inherent sensitivity and selectivity, CE-MS is a powerful technology for bio-analysis. In the novel field of application described herein, the contact of these two methods can lead to a better DNA sequence analysis method that outperforms the sequencing method of current ratio by some order of magnitude.
The fact that the correspondence between CE and Electrospray Ionization (ESI) flow rates and facilitated by (and primarily used for) the type of ions in solution provides the basis for highly effective formulations. The combination of both isotacophoresis using capillary region electrophoresis (CZE) and capillary quadrupole mass spectrometry based on ESI is described (Olivares et al., Anal. Chem. 59: 1230, 1987 Loo et al., Anal. Chem. 179: 404, 1989; Edmonds et al., J. Chroma. 474: 21, 1989; , J. Chromatog. 480: 211, 1989, Grese et al., J. Am. J. Microcolumn Sep. 1: 223, 1989; Lee et al., J. Chromatog. 458: 313, 1988; Smith et al. Chem. Soc. 111: 2835, 1989). Small peptides can be readily CZE analyzed with excellent (femto) sensitivity.
The most powerful separation method for DNA fragments is generally polyacrylamide gel electrophoresis (PAGE) in the form of slab gels. However, most of the limitations of current technology are that it takes a relatively long time to perform gel electrophoresis of the DNA fragments produced in the sequencing reaction. Can be accomplished using capillary electrophoresis using a very thin gel by increased multiplication (10 times). In the glass solution for the first approximation, all DNA moves to the same mobility as the mass and charge are compensated for by adding a base. For polyacrylamide gels, the DNA fragments are seeded and moved as a function of length, and this approach is applied to CE. Significant plate counts per meter are now achieved using crosslinked polyacrylamides (see 10 ⁺⁷ plates per meter, Cohen et al., Proc. Natl. Acad. Sci., USA 85: 9660, 1988). As described, these CE columns can be used for DNA sequencing. The CE method is, in principle, 25 times faster than slab gel electrophoresis in the standard cryostat. For example, about 300 bases can be read per hour. The separation rate is limited to slab gel electrophoresis by amplifying the electric field that can be applied to the gel without excessively forming heat. Thus, the faster speed of the CE is achieved by using a higher electric field strength (300 V / cm for CE versus 10 V / cm for slab gel electrophoresis). The shape of the capillary reduces the amount of current and power and reduces the formation of heat.
Smith et al., Nuc. Acide. Res. 18: 4417, 1990) suggests using multiple capillaries in parallel to increase throughput. Similarly, the literature (Mathies and Hung, Nature 359: 167, 1992) introduces capillary electrophoresis in which separation is performed on a parallel array of capillaries and high throughput sequencing is performed (Huang et al. Anal. Chem. 64: 967, 1992, Huang et al., Anal. Chem. 64: 2149, 1992). The main disadvantage of capillary electrophoresis is that the amount of sample that can be added to the capillary is limited. By concentrating a large amount of sample at the start of the capillary prior to separation, the load can be increased and the detection concentration can be reduced by several times. The most popular preconcentration method for CE is stacking of samples. Sample stacking has been reviewed recently (Chien and Vurgi, Anal. Chem. 64: 489A, 1992). Sample stacking between the sample buffer and the capillary buffer depends on the matrix deviation (pH, ionic strength), so that the electric field across the sample area is stronger than in the capillary region. For stacking the samples, a sample of large volume of low concentration buffer is introduced for preconcentration at the tip of the capillary column. The capillaries are charged with the same composition but with a higher buffer. When sample ions reach the capillary buffer and lower electric field, they are stacked into the dense area. The stacking of the samples increases the detection performance by 1 to 3 times.
Another method of preconcentration is to apply isotacophoresis (ITP) prior to separation of the free region CE of the analyte. ITP is an electrophoretic technique that allows loading micro-liter volumes of sample into a capillary in contrast to associating a low nL injection volume typically with CE. This technique relies on inserting the sample between two buffers (inducing and decreasing electrolysis) of higher mobility and lower mobility than analytes respectively. This technique is inherently an enrichment technique that concentrates the analyte into a pure region at the same rate. At present, this technique is less popular than the stacking method described above, because it requires some selection to induce and reduce electrolysis and requires separation of only the cationic or anionic material in the separation process.
A key aspect of the DNA sequencing process is the remarkably selective electrophoretic separation of DNA or oligonucleotide fragments. This is clear, for example, that each fragment is disassembled and only the nucleotide is different. A separation of up to 1000 fragments (1000 bp) is obtained. Additional advantages of sequencing using cleavable tack are as follows. When a cleavable tack is used, it is not necessary to use a slab gel form if the DNA fragment is separated by polyacrylamide gel electrophoresis. Since multiple samples are combined (4 to 2000), it is not necessary to perform the samples in parallel, such as when using a dye-primer or dye-terminator method (i.e., the ABI373 sequencer). There is no reason to use a slab gel because there is no reason to do parallel lanes. Thus, a tube gel form for the electrophoretic separation method can be used. (Grossman et al., Genet. Anal. Tech., Appl. 9: 9, 1992) show a considerable advantage obtained when the tube gel form is used instead of the slab gel form. This is due to the higher performance of the waste lining in tube form and the higher degradation of high molecular weight DNA fragments (1000 nt) compared to slab gels resulting in faster run times (up to 50%). Long reading is important for genome sequencing. Thus, the use of truncable taps in sequencing has the additional advantage of allowing the user to use the most effective and sensitive methods of DNA isolation that have the highest resolution.
4. Micro-assembled device
Capillary electrophoresis (CE) is a powerful method for DNA sequencing, forensic analysis, analysis of PCR products and sizing of restriction fragments. CE is much faster than conventional slap PAGE because capillary gel can be used to apply a much higher total stomach. However, CE has the disadvantage of allowing only one sample per gel to be processed. The method combines the faster separation time of CE and the ability to analyze multiple samples in parallel. An important concept underlying the use of microassembled devices is the ability to increase the information density in electrophoresis by maximizing the size of the lane to about 100 μm. The electronics industry typically manufactures circuits with characteristics less than 1 micron in size using microassembly. The current density of the capillary array is limited to the outer diameter of the capillary tube. Micro-assembly of the channels forms a high-density array. Microassembly also allows physical assembly that is not possible when using glass fibers and joins the channels directly to other devices on the chip. Few devices are configured on a microchip for separation technology. Liquid chromatography (Manz et al., Sens. Actuators B1: 249, 1990) and gas chromatography (Terry et al., IEEE Trans. Electron Device, ED-26: 1880, But these devices are not widely used. Several groups have reported isolated fluorescent dyes and amino acids on microassembled devices (Manz et al., J. Chromatography 593: 253, 1992, Effenhauser et al., Anal. Chem. 65: 2637, 1993). In recent years it has been shown to use optical lithography and chemical etching to produce multiple separation channels on a glass substrate (Woolley and Mathies, Proc. Natl. Acad. Sci. 91: 11348, 1994). The channel is filled with a hydroxyethyl cellulose (HEC) separation matrix. This indicates that DNA restriction fragments can be separated in a short time of about 2 minutes.
D. Cutting of tack
As described above, different linker forms can impart cutting performance (" unstable performance ") under different specific physical or chemical conditions. Examples of conditions used for cleaving linkers of various designs include acid, base, oxidation, reduction, fluorine, thiol exchange, photolysis, and conditions by enzymes.
Examples of cleavable linkers that meet the general standard for the linkers listed above are those well known to those skilled in the art and include those shown in the catalog available from Pierce, Rockford, IL. Examples of these include:
· Ethylene glycobis (succinimidyl stearate) (EGS), an amine reactive cross-linking agent that can be cleaved by hydroxylamine (1M at 37 ° C for 3 to 6 hours);
· Amine reactive crosslinking agents which can be cleaved by disuccinimidyl tartrate (DST) and sulfo-DST, 0.015 M sodium periodate
Amine-reactive crosslinking agents which can be cleaved by bis [2- (succinimidyloxycarbonyloxy) ethyl] sulfone (BSOCOES) and sulfo-BSOCOES, base (pH 11.6);
A pyridyldithiol cross-linking agent which can be cleaved by 1,4-di- [3 '- (2'-pyridyldithio (propionamido)) butane (DPDPB), thiol exchange or reduction;
- Pyridyl dithiols that can be cleaved by thionylation or reduction, such as N- [4- (p-azidosalicylamido) -butyl] -3 '- (2'-pyridyldithio An organic crosslinking agent;
Photoreactive crosslinking agents which can be cleaved by bis- [beta-4- (isosalicylamido) -ethyl] disulfide, thiol exchange or reduction;
Photoreactive crosslinking agents which can be cleaved by N-succinimidyl- (4-azidophenyl) -1,3'-dithiophosphonate (SADP), thiol exchange or reduction;
-Sulphosuccinimidyl-2- (7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3'-dithiophosphonate (SAED), cut by thiol exchange or reduction Photoreactive cross-linking agents;
· Photoreactive crosslinking which can be cleaved by sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido) -1,3'-dithiophosphonate (SAND), thiol exchange or reduction Binder.
Other examples of linkers that can be cut and the cutting conditions that can be used to release the tack are as follows. The silyl bonding groups can be cleaved by fluorine or under acidic conditions. The 3-, 4-, 5- or 6-substituted 2-nitrobenzyloxy or 2-, 3-, 5- or 6-substituted 4-nitrobenzyloxy bond group is cleaved by light source . 3-, 4-, 5-, or 6-substituted-2-alkoxyphenoxy or 2-, 3-, 5- or 6-substituted-4-alkoxyphenoxy bond group Ce (NH _{4) 2} (NO ₃ ) ₆ (oxidation). NCO ₂ (urethane) linkers can be cleaved by hydroxides (bases), acids or LiAlH ₄ (reduction). The 3-pentenyl, 2-butenyl or 1-butenyl bond group can be cleaved by O ₃ , O _s O ₄ / IO ₄ ^- , or KMnO ₄ (oxidation). The 2- [3-, 4- or 5-substituted-furyl] oxy bond group can be cleaved by O ₂ , Br ₂ , MeOH or an acid.
Other labile conditions for cleaving bond groups include: t-alkyloxy bond groups can be cleaved by an acid; Methyl (dialkyl) methoxy or 4-substituted-2-alkyl-1,3-dioxolane-2-yl bond group can be cleaved by H ₃ O ⁺ ; The 2-silylethoxy bond group may be cleaved by fluorine or an acid; 2- (X) -ethoxy, where X is a keto, ester amide, cyano, NO ₂ , sulfide, sulfoxide, sulfone bond group can be cleaved under alkaline conditions; The 2-, 3-, 4-, 5- or 6-substituted-benzyloxy bond group may be cleaved under acid or reducing conditions; The 2-butenyloxy linkage group can be cleaved by (Ph ₃ P) ₃ PhCl (H); The 3-, 4-, 5- or 6-substituted-2-bromophenoxy bond group may be cleaved by Li, Mg or BuLi; The methyl thiomethoxy bond group can be cleaved by Hg ²⁺ ; 2- (X) -ethyloxy (wherein X is a halogen) bond group may be cleaved by Zn or Mg; The 2-hydroxyethyloxy linkage group can be cleaved by oxidation (e.g., Pb (OAc) ₄ );
A preferred linker is a linker that is cleaved by acid or photolysis. Several acid-labile linkers that have been developed for solid phase peptide synthesis are useful for binding tack to MOI. Some of these linkers are described in a recent article by Lloyd-Wiliams et al. (Tetrahedron 49: 11065-1113, 1993). One useful form of linker is based on p-alkoxybenzyl alcohol and two 4-hydroxymethylphenoxyacetic acid and 4- (4-hydroxymethyl-3-methoxyphenoxy) butyric acid are commercially available [ Acvanced ChemTech (Louisville, KY). Both linkers can be attached to the tack via ester linkages to the benzyl alcohol and attached to the MOI containing amines via amide linkages to the carboxylic acid. The bonds bound by these molecules are released from the MOI with varying concentrations of trifluoroacetic acid. Carboxylic acid is released from the tack by the cleavage of these linkers. In a tack released by acid cleavage of a tack attached via an associated linker such as 2,4-dimethoxy-4 '- (carboxymethyloxy) -benzhydrylamine (manufacturer: AcmeChemTech, FMOC-protected form) To induce the release of the carboxylamide.
Also, photo-labile linkers useful in these applications have been developed for the synthesis of solid phase peptides (see Lloyd-Williams review). These linkers are typically based on 2-nitrobenzyl esters or 2-nitrobenzyl amides. Two examples of photolabile linkers recently reported in the literature are: 4- (4- (1-Fmoc-amino) ethyl) -2-methoxy-5-nitrophenoxy) butanoic acid (Holmes and Jones, J (Fmoc-amino) -3- (2-nitrophenyl) propionic acid (Brown et al., Molecular Diversity 1: 4-12, 1995) . These two linkers are attached to the amino in the MOI via a carboxylic acid. Attachment of the tack to the linker is accomplished by forming an amine between the carboxylic acid in the tack and the amine in the linker. The cleavage of the photolabile linker is typically performed using UV light at a wavelength of 350 nm at an intensity and time known in the art. Examples of manufacturers of devices for photochemical cleavage are Aura Industries Inc. (Staten Island, NY) and Agrenetics (Wilmington, WA). When the linker is cleaved, the amide of the free radicals is released first. Examples of photo-cleavable linkers include nitrophenyl glycine ester, exo- and endo-2-benzo norbornyl chloride and methanesulfonate and 3-amino-3 (2-nitrophenyl) propionic acid. Examples of cleavage by an enzyme include an esterase capable of cleaving an ester bond, a nuclease capable of cleaving a phosphodiester bond, a protease cleaving a peptide bond, and the like.
E. Detection of tack
Typically, the detection method relies on absorption and emission in some form of quenching. When an atom or molecule absorbs light, the energy introduced introduces the quantized structure into a higher energy level. The shape here depends on the wavelength of the light. The electrons are promoted to a higher orbit by ultraviolet or visible light, the molecular vibration is excited by infrared rays, and the rotation is excited by electromagnetic waves. The absorption spectrum is absorption of light as a function of wavelength. The spectrum of an atom or molecule depends on the structure of its energy level. Absorption spectra are useful for identification of compounds. Particular absorption spectroscopy methods include electron absorption spectroscopy (AA), infrared spectroscopy (IR) and UV-visible spectroscopy (uv-vis).
Atoms or molecules that are excited at high energy levels can decay to lower levels as they emit radiation. This emission of light is called fluorescence when there is a transition between the same spin states and phosphorescence when there is a transition between different spin states. The emission intensity of the analyte is linearly proportional to the concentration (at low concentrations) and is useful for quantizing the emitted material. Specific emission spectrometry methods include atomic emission spectroscopy (AES), atomic fluorescence spectroscopy (AFS), molecular laser induced fluorescence (LIF) and X-ray fluorescence (XRF).
When electromagnetic radiation passes through a material, most of the radiation continues in its original direction, but a small amount of the fragment is scattered in the other direction. The light scattered as the introduced light at the same wavelength is called Rayleigh scattering. Light that is scattered in a transparent solid due to vibration (acoustic quantum) is called Brillouin scattering. Brillouin scattering typically travels from the incident light by 0.1 to 1 wave number. In opaque solids, light scattering due to vibration in both molecules or optical acoustics is called Raman scattering. The light that is scattered by the rays travels 4000 waves from the incident light. Specific scattering spectrometry methods include racane spectroscopy.
IR spectroscopy analyzes the absorption wavelength and intensity of the medium-infrared light by the sample. Medium-infrared (2.5 to 50 μm, 4000 to 200 cm ^-1 ) is strong enough to excite molecular vibrations to higher energy levels. The wavelength of the IR absorption band is specific to a particular type of chemical bond and the IR spectrum is generally most useful for identification of organic and organometallic molecules.
Near-infrared absorption spectroscopy (NIR) measures the absorption wavelength and intensity of near-infrared light by a sample. The mid-infrared range is in the range of 800 nm to 2.5 占 퐉 (12,500 to 4000 cm ^-1 ) and is strong enough to excite the correspondence and combination of molecular vibrations to higher energy levels. NIR spectroscopy is typically used for quantitative determination of organic functional groups, especially OH, NH and C = O. The composition and design of the NIR instrument is similar to the uv-vis absorption spectrometer. The light source is typically a tungsten lamp and the detector is typically a PbS solid-state detector. Sample holding group is free or can be quartz and typical solvents are CCl ₄ and CS _2. It is suitable for on-line monitoring and process control by convenient mechanism operation of NIR spectroscopy.
Ultraviolet and visible absorption spectroscopy (uv-vis = Ultraviolet and Visible Absorption Spectroscopy) measures the absorption wavelength and intensity of near-ultraviolet and visible light by a sample. Adsorption at vacuum UV takes place at 100 to 200 nm; (10 ⁵ to 50,000 cm ^-1 ) quartz UV at 200 to 350 nm; (50,000 to 28,570 cm <" ¹ >) and visible light at 350 to 800 nm; (28,570 to 12,500 cm ^-1 ) Beer-Lambert-Bouguet law. Ultraviolet and visible light are powerful enough to promote external electrons to higher energy levels. The uv-vis spectroscopic analysis is typically applied to complexes of molecules and inorganic ions or in solution. The uv-vis spectrum is limited by the broad spectrum of the spectrum. The light source is typically a hydrogen or deuterium lamp for uv measurements and a tungsten lamp for visible light measurements. The wavelength of such a continuous light source is selected using a wavelength separator such as a prism or a grating monochromator. The spectrum is obtained by scanning the wavelength separator and the quantitative measurement can be made from the spectrum or at a single wavelength.
Mass spectrometers use deviations in mass to charge ratio (m / z) of ionizing atoms or molecules, respectively, to separate from each other. Mass spectrometry is useful for determining the amount of atoms or molecules and is useful for determining chemical and structural information about a molecule. The molecule has a characteristic fragmentation pattern that provides structural information identifying the identity of the compound. The general operation of mass spectrometry is as follows. Gaseous phase ions are formed and ions are separated in space or time based on their mass to charge ratios and the amount of ions in each mass to charge ratio is measured. The ion dissociation power of the mass spectrometry analysis is described by the resolution defined as R = m / delta m, where m is the ion mass and delta m is the deviation in mass between the two resolvable peaks in the mass spectrum. For example, a mass spectrometer having a resolution of 1000 can decompose ions having an m / z of 100.0 from an ion having an m / z of 100.1.
Generally, a mass spectrometer (MS) consists of an ion source, a mass selective analyzer and an ion detector. The magnetic sector, quadrupole, and time-of-flight design also require extension and accelerated ion optics to move ions from the source region to the mass spectrometer. Details of some mass analyzer designs (e.g., magnetic sector MS, quadrupole MS or flight time MS) are discussed below. A single focusing analyzer for the magnetic sector MS uses particle beam path 180, 90 or 60 degrees. Various forces affect particle separation ions with different mass to charge ratios. An electrostatic analyzer is attached to this type of instrument using a double focusing analyzer to separate particles with different dynamic energies.
Quadrupole The quadrupole mass filter for MS consists of four metal rods arranged in parallel. The applied voltage affects the trajectory of ion movement down the flight path formed between the four rods. For a given DC and AC voltage, ions of a specific mass to charge ratio through the quadrupole filter and all other ions deviate from their original path. The mass spectrum is obtained by monitoring the ions passing through the quadrupole filter as the voltage on the rod changes.
Time-of-flight mass spectrometers use different deviations in travel time through a " drift region " to separate ions of different masses. It must operate in a pulsed mode to produce ions from pulses and / or extract them from pulses. The pulsed electric field accelerates all ions to the long-glass drift region with dynamic energy qV, where q is the ionic charge and V is the applied voltage. Because the kinetic energy of the ions is 0.5 mV ² , the lighter ions have a higher voltage than the heavier ions and reach the detector at the end of the drift region more quickly. The output of the ion detector is displayed on the oscilloscope as a function of time to form the mass spectrum.
The ion formation process is the starting point for mass spectrometry. Chemical ionization is a method of forming ions by proton or hybrid migration using a preparation ion that reacts with a molecule (tag) of the analyte. The formulation ions are prepared by introducing excess methane (for tack) into the electron impact (EI) ion source. The electron collision reacts further with methane to produce CH ₄ ⁺ and CH ₃ ⁺ which form CH ₅ ⁺ and C ₂ H ₅ ⁺ . Another method of ionizing the tack is by plasma and glow discharge. Plasma is a hot, partially ionized gas that effectively excites and ionizes atoms. The glow discharge is a low-pressure plasma held between two electrodes. Electronic impact ionization ionizes gaseous atoms or molecules using an electron beam, which is typically produced from tungsten filaments. Electrons from the beam break electrons into atoms or molecules of the analyte to form ions. Electrospray ionization uses very fine needles and a series of skimmers. The sample solution is sprayed into the source chamber to form a droplet. The droplet holds charge when the outlet is a capillary and as the solvent evaporates, the droplet disappears while leaving the molecule of the highly charged analyte. ESI is useful for large biological molecules that are difficult to evaporate or ionize. Fast atom bombardment (FAB) uses a high energy beam of neutral atoms, typically Xe or Ar, which collide with a solid sample to cause desorption and ionization. It is used for large biological molecules that are difficult to form a gas phase. FAB is almost free from fragmentation and is usually useful for molecular weight determination by providing ionic peaks of large molecules. An atomic beam is produced by accelerating ions from an ion source through a charge exchange cell. Ions select electrons in collisions with neutral atoms to form a beam of high energy atoms. Laser ionization (LIMS) is a method in which a laser pulse forms a microplasma that removes material from the surface of the sample and ionizes a portion of the sample constituents. Matrix-assisted laser desorption ionization (MALDI) is a LIMS method of evaporating or ionizing large biological molecules such as proteins or DNA fragments. Biological molecules are distributed in a solid matrix such as nicotinic acid. The UV laser pulse removes a matrix that can be extracted into a mass spectrometer by moving some large molecules into ionized form in the gas phase. Plasma-desorption ionization (PD) utilizes a decay of ²⁵² Cf to produce two fission fragments moving in opposite directions. One fragment collides a sample falling into 1 to 10 analyte ions. Other fragments collide the detector and trigger the start of data acquisition. Such ionization methods are particularly useful for large biological molecules. Resonance ionization (RIMS) is a method in which more than one beam is accelerated in a stepwise manner above the ionization potential to form gaseous atoms or molecules in resonance to form ions. Secondary ionization (SIMS) uses ion beams such as ³ He ⁺ , ¹⁶ O ⁺ , or ⁴⁰ Ar ⁺ ; Focus on the surface of the sample and sputter the material in gas phase. A spark source is a method of ionizing an analyte by pulsing a current across two electrodes in a solid sample.
The tack can be charged before, during, or after cutting from the attached molecule. Ionization methods based on ion " desorption ", direct formation or release of ions from a solid or liquid surface allows for increased application to non-volatile and thermally labile compounds. This approach eliminates the need to volatilize neutral molecules prior to ionization and generally resolves thermal denaturation of molecular materials. These methods are described in Bedky, Principles of Field Ionization and Field Desorption Mass Spectrometry (Pergamon, Oxford, 1977), plasma desorption (see Sunkqvist and Macfarlane, Mass Spectrom. Rev. 4: 421, 1985) (See, for example, fast atom bombardment, FAB, and (see, for example, Karl and Hillenkamp, Anal. Chem. 60: 2299, 1988; Karas et al., Angew. Chem. Includes thermal ionization (TS) and ionization (see Vestal, Mass Spectrom. Rev. 2: 447, 1983), secondary ion mass spectrometer (SIMS, Barber et al., Anal. Chem. 54: 645A, 1982) do. Thermal dispersion is widely applied with liquid chromatography for on-line compounding. Continuous flow FAB methods (see Caprioli et al., Anal Chem. 58: 2949, 1986) also exhibit significant potential. A more complete list of ionization / mass spectrometry assays is ion-trap mass spectrometry, electrospray ionization mass spectrometry, ion spray mass spectrometry, liquid ionization mass spectrometry, atmospheric pressure ionization mass spectrometry, electron ionization mass spectrometry, Stability atomic impact ionization mass spectrometry, fast atomic impact ionization mass spectrometry, MALDI mass spectrometry, photo-ionization time-of-flight mass spectrometry, laser dot mass spectrometry, MALDI-TOF mass spectrometry, APCI mass spectrometry, - Spray mass spectrometry, nebulization atomization mass spectrometry, chemical ionization mass spectrometry, resonance ionization mass spectrometry, secondary ionization mass spectrometry, and thermal spray mass spectrometry.
Ionization methods are possible for non-volatile biological compounds that encompass a range of applicability. The ionization efficiency is highly dependent on the matrix composition and the form of the compound. Currently available results indicate that the upper molecular weight limit for TS is about 800 dalton (Jones and Krolik, Rapid Comm. Mass Spectrom. 1:67, 1987). Sensitivity is typically irregular at a higher mass to charge ratio (m / z), since TS is typically tested using a quadrupole mass spectrometer. Time-of-flight (TOF) mass spectrometers are commercially available and have the advantage that the range of m / z is limited only by the efficiency of the detector. Recently, two additional ionization methods have been introduced. These two methods are currently used for matrix atomic laser desorption (MALDI, Karas and Hillenkamp, Anal. Chem. 60: 2299, 1988; Karas et al., Angew. Chem. 101: 805, 1989) . Both methods have very high ionization efficiencies (i.e., very high [repared molecular ion] / [consumed molecule]). The sensitivity that defines the final performance of the technique depends on the size of the sample, the amount of ions, the flow rate, the detection efficiency, and the actual ionization efficiency.
Electrospray-MS is based on the idea first proposed in the 1960s (Dole et al., J. Chem. Phys. 49: 2240, 1968). Electrospray ionization (ESI) is one means of preparing charged molecules for analysis by mass spectrometry. Simply electrospray ionization produces highly charged spots by spraying the liquid in a strong electrostatic field. Generally, highly charged dots formed in the gas of anhydrous bath at atmospheric pressure are contracted by evaporation of the neutral solvent until the repulsion of the charge overcomes cohesion and causes a "Coulomb explosion". The exact mechanism of ionization is debatable and some groups provide hypotheses (see Blakes et al., Anal. Chem. 63: 2109-14, 1991; Kebarle et al., Anal. Chem. -86, 1993; Fenn, J. Am. Soc. Mass. Spectrom. 4: 524-35, 1993). Regardless of the ultimate process of ion formation, ESI produces charged molecules from solution under mild conditions.
The ability to obtain useful mass spectra for small amounts of organic molecules depends on the effective production of ions. The efficiency of ionization for ESI is related to the degree of positive charge associated with the molecule. Improving experimental ionization typically involves the use of acidic conditions. Another way to improve ionization is to use quaternary amines where possible (see Aebersold et al., Protein Science 1: 494-503, 1992; Smith et al., Anal. Chem. 60: 436-41 , 1988).
Electrospray ionization is described in more detail below. Electrospray ion formation requires two steps: near atmospheric pressure and then dispersing highly charged drops at conditions that lead to evaporation. The solution of the analyte molecule is passed through a needle held on a high electric bottom. At the end of the needle, the solution is dispersed into a small, highly charged dot containing the analyte molecule. Small amounts of droplets evaporate quickly and protein molecules quantitated by residual evaporation are released into the gas phase. Electrospray is generally produced by applying a high electric field from a capillary to a small amount of flowing liquid (typically 1 to 10 [mu] L / min). A potential difference of 3 to 6 kV is typically applied between the capillary and the counter electrode located 0.2 to 2 cm apart where ions, charged clusters and even charged dots, depending on the degree of desolvation, are removed by the MS via a small orifice Can be sampled). The electric field causes the aggregation of charge on the liquid surface at the end of the capillary; The liquid flow rate, resistance, and surface tension are therefore important factors in the manufacture of the drop. High electric fields cause disturbance of the liquid surface and formation of highly charged liquid droplets. Positive or negative charged dots can be produced depending on the bias of the capillary. The negative ion mode requires the presence of an electronic scavenger such as oxygen to suppress electrical discharge.
A wide range of liquids can be vestibrated in the vacuum or with the aid of a spray. The use of an electric field only for spraying results in some practical limitations on the range of liquid charge capacity and dielectric constant. The filling capacity of the solution, 10 ^-5 ohms, is required for stable atomization at a useful liquid flow rate corresponding to less than 10 ^-4 M aqueous electrolytic solution at room temperature. In the mode most usefully found for ESI-MS, a suitable liquid flow rate causes the dispersion of the liquid as fine mist. At short distances from the capillary the spot diameter is often very uniform and about 1 micrometer. It is particularly important that the overall electrospray ion slightly increases the current only for a higher liquid flow rate. Heating has proven useful in amplifying electrospray. For example, some heating may cause the aqueous solution to be rapidly atomized due to reduced viscosity and surface tension. Thermal assist and gas-atom-assisted electrospray enable higher liquid flow rates, but reduce the degree of dot charge. In order to form a molecular ion, conditions for performing initial evaporation of the tap water are required. This can be achieved by heating during movement through the interface at moderate temperatures (less than 60 ° C) by flow of a stream of gas at high pressure and by strong collisions at relatively low pressures (especially in the case of ion trap methods).
Even though the details of the process remain unclear under the ESI, a very small drop produced by ESI appears to allow almost any material bearing a net charge in the solution to migrate to the gaseous phase after evaporation of the residual solvent. The mass spectrometry detection then requires the ion to have a m / z range (less than 4000 dalton for a quadrupole instrument) that is operable after desolvation, is manufactured and needs to travel with sufficient efficiency. The lack of dependence of the ionization efficiency on a wide range of eluent and virtually molecular weight found to be feasible for ESI-MS suggests a highly non-discriminatory and widely applicable ionization process.
Electrospray ion " source " functions near atmospheric pressure. An electrospray " source " is typically a metal or glass capillary that incorporates a method for electrically biasing a liquid solution against a counter electrode. The solution, which is a water-methanol mixture containing analyte and often other additives such as acetic acid, flows to the capillary end. The ESI source is described in the literature (Smith et al., Anal Chem. 62: 885, 1990) and specifically replenishes any solvent system. A typical flow rate for ESI is 1 to 10 [mu] L / min. An important requirement of the ESI-MS interface is to sample and transfer ions into the MS as efficiently as possible from the high-pressure region.
The efficiency of ESI is very high and provides the basis for highly sensitive measurements useful in the present invention described herein. Current instruments can be used to provide a total ion current of about 2 x 10 ^-12 A or 10 ⁷ counts / s for a single charged material in the detector. Based on the performance of the instrument, the concentration of a single charged material of 10 ^-10 M or about 10 ^-18 mol / s can provide detectable ionic current (about 10 counts / s) when the analyte is fully ionized have. For example, low atomic molar detection limits are obtained for quaternary ammonium ions using an ESI interface using capillary domain electrophoresis (Smith et al., Anal. Chem. 59: 1230, 1988). For a compound with a molecular weight of 1000, the average number of charges is 1, the approximate number of charge states is 1, the peak width (m / z) is 1 and the maximum intensity (ions / s) is 1 x 10 ¹² .
Remarkably, in obtaining the ESI mass spectra the sample is virtually depleted (see Smith et al., Anal. Chem. 60: 1948, 1988). Can in fact be obtained by the use of an array detector having a sector mechanism that allows simultaneous detection of a portion of the spectrum. Since all of the ions currently formed by ESI, about 10 < ^-5 > are detected, provide a basis for improved sensitivity by focusing on factors limiting the performance of the apparatus. This will be apparent to those skilled in the art with reference to the present invention and to improve on ionization and detection methods.
The interface can preferably be located between a separation mechanism (e. G., A gel) and a detector (e. G., A mass spectrometer). The interface preferably has the following characteristics: (1) the ability to collect DNA fragments at prudent time intervals, (2) the ability to concentrate the DNA fragments, (3) the ability to remove DNA fragments from electrophoresis buffers and environments (5) ability to separate tags from DNA fragments; (6) ability to distribute DNA fragments; (7) ability to place tags in volatile solutions; (8) ) The ability to volatilize and ionize tack, and (9) the ability to place tacks in an electrospray and transfer them to introduce tack in mass spectrometric analysis.
In addition, the interface has the ability to " collect " DNA fragments as they elute from the bottom of the gel. The gel may consist of slab gel, tubular gel, or capillary. DNA fragments can be collected by several methods. The first method uses an electric field, where DNA fragments are collected on or near the electrodes. The second embodiment is a method in which the DNA fragments are collected by flowing the liquid stream to the bottom of the gel. Both modes of embodiment can be combined and the DNA collected into a flow stream that can be concentrated using a subsequent electric field. The end result is to remove the DNA fragment from the environment in which the separation method is performed. That is, DNA fragments can be transferred from one solution form to another using an electric field.
Once a DNA fragment is present in a suitable solution (which may be in parallel with electrophoresis and mass spectrometry), the tack can be cleaved from the DNA fragment. The DNA fragment (or its residues) can be separated from the tag by applying an electric field (preferably the tag is the opposite charge of the charge of the DNA tag). The tack is then introduced into the electrospray device using an electric field or a flowing liquid.
Most of them can identify and quantify the stagnation of the fluorescent tag directly by their absorption and fluorescence emission wavelength and intensity.
Conventional spectrophotometers that provide a continuous range of excitation and emission wavelengths ( _EX , I _S1, I _S2 ) are highly variable, while more specialized instruments such as flow cytometers and laser scanning microscopes are used at a single fixed wavelength It needs probes that can be excited. In a replenishing mechanism, this is typically a 488-nm line of argon laser.
The fluorescence intensity per probe molecule is proportional to the product of e and QY. In fact, among these presently important fluorophore groups, the range of these parameters is about 10,000 to 100,000 cm ^-1 M ^-1 for ε and 0.1 to 1.0 for QY. When the absorption induces saturation by high-intensity illumination, the irreversible degeneration (excitation) of the excitation fluorophores becomes a factor limiting the fluorescence detection performance. The actual effect of Kwon Baebak depends on the fluorescence detection technique in question.
It will be apparent to those skilled in the art that one device (one interface) is located between the separation and detection steps to permit continuous manipulation (actual time) of size separation and detection of tack. This associates a mechanism with a separation method and a detection method and a mechanism that forms a single apparatus. For example, the interface is located between the separation technique and detection by mass spectrometry or constant electric current method.
The function of the interface is primarily to release the tag (e.g., mass spectrometry) from the analyte. There are several representative tools at the interface. The design of the interface depends on the choice of severable linker. For light-excitable linkers, an energy or quantum source is needed. In the case of acid labile linkers, base labile linkers or disulfide linkers, it is necessary to add a formulation between the interfaces. For thermal instability linkers, a thermal energy source is required. It is necessary to add enzymes to enzyme-sensitive linkers such as specific protease and peptide linkers, nuclease and DNA or RNA linkers, glycosylase, HRP or phosphatase, and an unstable linker after cleavage (e. G. Do. Other features of the interface include minimal band amplification, separation of DNA from the tag prior to injection into the mass spectrometer. Separation techniques include techniques based on electrophoresis methods and techniques such as size maintenance (dialysis), filtration, and the like.
In addition, the tack (or nucleic acid-linker-tack structure) can be concentrated and released by electrophoresis and into other product streams compatible with the particular type of ionization method selected. In addition, the interface can capture the tack (or nucleic acid-linker-tack structure) on the microbead and place the bead (s) into the chamber and perform laser desorption / evaporation. It can also be flowed and extracted into another buffer (e.g., from the capillary electrophoresis buffer across the permeation membrane into the hydrophobic buffer). Also, for some applications it may be desirable to immediately move the tack to a mass spectrometer, which may include the additional function of the interface. Another function of the interface is to transport the tag from multiple columns to the mass spectrometer with slots of rotation time for each column. The tack can also be carried from a single column to multiple MS detectors and separated by time, and each set of taps can be collected for several milliseconds and carried to a mass spectrometer.
The following is a list of typical practitioner of the separation technique and detection technologies which may be used herein: to prepare an electrophoresis device (Two Step ^TM, Poker Face ^TM II) for application to the sequencing [roducer: Hoefer Scientific Instruments (San Francisco, CA). (Pharmacia Biotech (Pescataway, NJ)) to prepare an electrophoresis apparatus (PhastSystem for PDR-SSCP analysis, DNA microarray for DNA sequencing and DNA sequencing). (ABI 373 and ABI 377) based on a fluorescence-dye (manufactured by Perkin Elmer / Applied Biosystems Division (ABI, Foster City, Calif.)). A UV spectrometer is manufactured [manufactured by Analytical Spectral Devices (Boulder, CO)]. An auto absorption spectrometer, a fluorescence spectrometer, an LC and GC mass spectrometer, an MNR spectrometer and a UV-VIS mass spectrometer are manufactured [Hitachi Instruments (Tokyo, Japan)]. Mass spectrometer is manufactured (PerSeptive Biosystems (Framingham, Mass.)). FTIR Spectrometer (Vector 22), FT-Raman Spectrometer, Time-of-Flight Mass Spectrometer (Reflex II ^TM ), Ion Trap Mass Spectrometer (Esquire ^TM ) and Maldi Mass Spectrometer [manufactured by Bruker Instruments INC. Manning Park, MA). A capillary gel electrophoresis unit, a UV detector, and a diode array detector are manufactured (Analytical Technology Inc (ATI, Boston, Mass.)). Ion trap mass spectrometers (3DQ Discovery ^TM and 3DQ Apogee ^TM ) (manufactured by Teledyne Electronic Technologies (Moutain View, Calif.)). A Sciex mass spectrometer (triplet LC / MS / MS, API 100/300) with compatibility with electrospray was prepared (Perkin Elmer / Applied Biosystems Division, Foster City, CA). A UV spectrometer as well as a mass selective detector (HP 5972A), a MALDI-TOF mass spectrometer (HP G2025A), a diode array detector, a CE unit, a HPLC unit (HP1090) as well as a Hewlett-Packard )]. Mass spectrometer (MAT 95 S ^TM , quadrupole spectrometer MAT 95 S ^TM and four other associated mass spectrometers) manufactured by Finigan Corporation (San Jose, Calif.). The HPLC apparatus is manufactured [manufacturer: Rainin (Emeryville, Calif.)].
The methods and compositions described herein allow for the use of truncated tact forms that serve as a map for specific sample shapes and nucleotide stagnation. The specific (selected) primers at the start of each sequencing method are indicated by a special tag. Forms a tack map for the form of the sample, the form of the dideoxy terminator (in the case of a luminescent sequencing reaction) or preferably both. In particular, the tack forms a map for primer types that form a map for a vector form that, in turn, forms a map for the identity of the sample. The tack can also map against the dideoxy terminator forms (ddTTP, ddCTP, ddGTP, ddATP) with reference to the primer set in the dideoxy reaction as the position sheet. After performing the sequencing reaction, the produced fragments are separated by size at the correct time.
The tag is truncated from the fragment in the temporary frame and recorded in the temporary frame. The sequence is constituted by a comparison of the tax map to the temporary frame. That is, the identity of all tacks is recorded after the sizing step at the correct time and correlated with each other in the temporary frame. The sizing step separates the nucleic acid fragments by one nucleotide amplification and consequently the identity of the related tack is separated by one nucleotide amplification. The sequence is quickly inferred in a linear fashion by prior knowledge of the dideoxy-terminal terminator or nucleotide map and the morphology of the sample.
The following embodiments are provided by way of example, not by way of limitation.
Unless otherwise stated, the chemicals as used in the examples can be obtained from the manufacturer (Aldrich Chemical Company, Milwaukee, Wis.). The following abbreviations are used:
ANP = 3- (Fmoc-amino) -3- (2-nitrophenyl) propionic acid
NBA = 4- (Fmoc-aminomethyl) -3-nitrobenzoic acid
HATU = O-7-amibenzotriazol-1-yl-N, N, N ', N'- tetramethyluronium hexafluorophosphate
DIEA = diisopropylethylamine
MCT = monochlorotriazine
NMM = 4-methylmorpholine
NMP = N-methylpyrrolidone
ACT357 = ACT357 peptide synthesizer (manufacturer: Advanced Chem Tech, Inc., Louisville, KY)
ACT = Advanced Chem Tech, Inc., Louisville, KY
NovaBiochem = CalBiochem-NovaBiochem International, San Diego, CA
TFA = trifluoroacetic acid
Tfa = trifluoroacetyl
iNIP = N-methylisophosphoric acid
Tfp = tetrafluorophenyl
DIAEA = 2- (diisopropylamino) ethylamine
MCT = monochlorotriazene
5'-AH-ODN = 5'-aminohexyl-tailed oligodeoxynucleotides
Example 1
Preparation of acid-labile linkers for use in sequencing-MW-identity determinants capable of cleavage.
A. Synthesis of a pentafluorophenyl ester of a chemically cleavable mass spectrometry tack for the release of a tack using the carboxylamide end.
Figure 1 shows the reactivity.
Step A
TentaGel S AC resin (Compound II; manufacturer: ACT; 1 equivalent) is suspended in a collection vessel of ACT357 peptide synthesizer (ACT) with DMF. Compound I (3 eq), HATU (3 eq) and DIEA (7.5 eq) in DMF are added and the collection vessel is shaken for 1 h. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling and washing steps for resin I are repeated, yielding compound III.
Step B
The resin (Compound III) is mixed with 25% piperidine in DMF and shaken for 5 minutes. The resin is filtered and then mixed with 25% piperidine in DMF and shaken for 10 minutes. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X) and DMF (2X) and used directly in Step C.
Step C
The deprotected resin from Step B is suspended in DMF and the amine function in DMF is added to the side chain (compound IV, e.g., alpha -N-FMOC-3- (3-pyridyl) -alanine, manufactured by Synthetech, Albany, HATU (3 eq.) And DIEA (7.5 eq.) In DMF (3 eq. The container is shaken for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X), and DMF (2X). The coupling and washing steps of IV to the resin are repeated to give compound V.
Step D
The resin (Compound V) is treated with piperidine as described in Step B to remove the FMOC group. The deprotected resin is then equally distributed from the collection vessel to the 16 reaction vessels by ACT357.
Step E
Sixteen aliquots of the deprotected resin from Step D are suspended in DMF. To each reaction vessel is added the appropriate carboxylic acid VI _1-16 (R _1-16 CO ₂ H; 3 equivalents), HATU (3 equivalents) and DIEA (7.5 equivalents) in DMF. The container is shaken for 1 hour. The solvent is removed and an aliquot of the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling and washing steps of VI _1-16 to aliquots of resin are repeated to give compounds VII _1-16 .
Step F
An aliquot of the resin (Compound VII _1-16 ) is washed with CH ₂ Cl ₂ (3X). To each reaction vessel is added 1% TFA in CH ₂ Cl ₂ and the vessel is shaken for 30 minutes. The solvent is filtered from the reaction vessel into individual tubes. An aliquot of the resin is washed with CH ₂ Cl ₂ ( ₂ ×) and MeOH (2 ×), and the filter is combined into individual tubes. The individual tubes are evaporated in vacuo to form compounds VIII _1-16 .
Step G
Each free carboxylic acid VIII _1-16 is dissolved in DMF. To each solution was added pyridine (1.05 eq.) And pentafluorophenyltrifluoroacetate (1.1 eq.) Was added. The mixture is stirred for 45 minutes at room temperature. The solution was diluted with EtOAc and a 1M ^- washed with aqueous citric acid (3X) and 5% aqueous NaHCO ₃ (3X), dried over Na ₂ SO _4, filtered, and evaporated in vacuo to compound Ⅸ _1-16 .
B. Synthesis of a pentafluorophenyl ester of a chemically cleavable mass spectrometry tack for the release of a tack using a carboxylic acid end.
Figure 2 shows the reactivity.
Step A
Compound 1 (4- (hydroxymethyl) phenoxybutyric acid) is combined with DIEA (2.1 eq) and allylobromide (2.1 eq.) In CHCl ₃ and heated to reflux for 2 h. The compound was diluted with EtOAc and washed with 1 N HCl (2X), pH 9.5 carbon buffer (2X) and brine (1X), dried over Na ₂ SO ₄ and evaporated in vacuo to give the allyl ester of Compound I do.
Step B
The allyl ester (1.75 eq.) Of Compound I from Step A was dissolved in CH ₂ Cl _{2 with} its side chain (Compound II, for example alpha-N-FMOC-3- (3-pyridyl) -alanine: manufacturer Synthetech, Albany , OR; 1 eq.) Is combined with an FMOC-protected amino acid containing an amine function, N-methylmorpholine (2.5 eq.) And HATU (1.1 eq.) And stirred at room temperature for 4 hours. The compound is diluted with CH ₂ Cl ₂ and washed with 1 M aqueous citric acid (2 ×), ANF (1 ×) and 5% aqueous NaHCO ₃ (2 ×), dried over Na ₂ SO ₄ and evaporated in vacuo. Compound III is liberated by flash chromatography (CH ₂ Cl ₂ -> EtOAc).
Step C
Compound III is dissolved in CH ₂ Cl ₂ , Pd (PPh ₃ ) ₄ (0.07 eq.), N-methyllaniline (2 eq.) Is added and the compound is stirred at room temperature for 4 hours. The compound is diluted with CH ₂ Cl ₂ , washed with 1 M aqueous citric acid (2 ×) and water (1 ×), dried over Na ₂ SO ₄ and evaporated in vacuo. Compound IV is liberated by flash chromatography (CH ₂ Cl ₂ -> EtOAc + HOAc).
Step D
Tentagel S AC resin (Compound V; 1 equivalent) is suspended in a collection vessel of ACT357 peptide synthesizer (manufacturer: Advanced ChemTech Inc. (ACT), Louisville, KY) with DMF. Compound IV (3 eq), HATU (3 eq) and DIEA (7.5 eq) in DMF are added and the collection vessel is shaken for 1 h. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling and washing steps for resin IV are repeated to give compound IV.
Step E
The resin (Compound IV) is mixed with 25% piperidine in DMF and shaken for 5 minutes. The resin is filtered and mixed with 25% piperidine in DMF and shaken for 10 minutes. The solvent is removed, the resin is washed with NMP (2X), MeOH (2X) and DMF (2X) and the deprotected resin is equally separated from the collection vessel to the 16 reaction vessel by ACT357.
Step F
Sixteen aliquots of the deprotected resin obtained from Step E are suspended in DMF. To each reaction vessel is added the appropriate carboxylic acid VIII _1-16 (R _1-16 CO ₂ H; 3 equiv.), HATU (3 equiv.) And DIEA (7.5 equiv.) In DMF. The container is shaken for 1 hour. The solvent is removed and an aliquot of the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling and washing steps of VII _1-16 to an aliquot of resin are repeated to give compounds VIII _1-16 .
Step G
An aliquot of the resin (Compound VIII _1-16 ) is washed with CH ₂ Cl ₂ (3X). To each reaction vessel is added 1% TFA in CH ₂ Cl ₂ and the vessel is shaken for 30 minutes. The solvent is filtered from the reaction vessel into individual tubes. An aliquot of the resin is washed with CH ₂ Cl ₂ ( ₂ ×) and MeOH (2 ×), and the filter is combined into individual tubes. The individual tubes are evaporated in vacuo to form compounds IX _1-16 .
Step H
Each of the free carboxylic acids IX _1-16 is dissolved in DMF. To each solution was added pyridine (1.05 eq.) And pentafluorophenyltrifluoroacetate (1.1 eq.) Was added. The mixture is stirred for 45 minutes at room temperature. The solution was diluted with EtOAc, washed with aqueous citric acid (3X) and 5% aqueous NaHCO ₃ (3X) of 1M, dried over Na ₂ SO _4, filtered, and evaporated in vacuo to a compound Ⅹ _1-16 .
Example 2
Explanation of cutting by photolysis of T-L-X
The T-L-X compound prepared according to Example 11 is irradiated with near UV light at room temperature for 7 minutes. A Rayonett fluorescent UV lamp (manufacturer: Southern New England Ultraviolet Co., Middletown, CT) having an emission peak at 350 nm is used as the UV light source. Place the lamp at a distance of 15 cm from the petri dish of the sample. SDS gel electrophoresis indicates that over 85% of the junctions were cleaved under these conditions.
Example 3
Preparation of fluorescently labeled primers and demonstration of cleavage of fluorophore
Synthesis and Purification of Oligonucleotides
Oligonucleotides (ODNs) are prepared in an automated DNA synthesizer using standard phosphoramidite chemistry or H-phosphonate chemistry (manufacturer: Glenn Research Sterling, VA) supplied commercially. Properly blocked dA, dG, dC and T phosphoramidites are commercially available in this form, and the synthetic nucleosides can be readily converted to other suitable forms. Oligonucleotides are prepared using commercially available standard phosphoramidite chemistry or H-phosphonate chemistry. Oligonucleotides are purified by the application of standard methods. 5'-trityl oligonucleotide having the group 12㎛, # 300 to nucleotide la coix seed;: HPLC and 0.1N using the (Rainin producer ^{_{Emeryville, CA) Et 3 NH +}} OAc - of the 15% to 55% MeCN Dynamax C-8 on a reversed phase column of 4.2x250 mm using a gradient elution buffer (pH 7.0) over 20 minutes. When the tritylation is carried out, the oligonucleotides are further purified by gel exclusion chromatography. Assays for the quality of oligonucleotides are performed by PRP-column (manufacturer: Alltech, Deerfield, IL) and PAGE at alkaline pH.
Preparation of 2,4,6-trichlorotriazine-derived oligonucleotides: 10-1000 μg of 5'-terminal terminated amine-linked oligonucleotides were dissolved in 10% excess recrystallized cyanuric acid in n-methyl-pyrrolidone And reacted in chloride and alkaline (preferably pH 8.3 to 8.5) buffer at 19 DEG C to 25 DEG C for 30 minutes to 120 minutes. The final reaction conditions consisted of 0.15 M sodium borate, 2 mg / ml recrystallized cyanuric acid chloride at pH 8.3 and 500 ug / ml of individual oligonucleotides. The unreacted cyanuric acid chloride is removed by size exclusion chromatography on a G-50 Sephadex (manufacturer: Pharmacia, Piscataway, NJ) column.
The active purified oligonucleotide is then reacted with 100 times molar excess cystamine in 0.15M sodium borate at pH 8.3 for 1 hour at room temperature. The unreacted cystamine is removed by size exclusion chromatography on a G-50 Sephadex column. The resulting ODN is then reacted with an amine-reactive fluorescent dye. The induced ODN preparation was dispensed into three fractions and each fragment was diluted with (a) 20 fold molar excess of Texas Red sulfonyl chloride (manufacturer: Molecular Probes, Eugene, OR), (b) lysamine ) 20 fold molar excess of sulfonyl chloride (manufacturer: Molecular Probes, Eugene, OR), and (c) 20 fold molar excess of fluorene isocyanate. The final reaction conditions consist of 0.15M sodium borate at room temperature for 1 hour at pH 8.3. The unreacted fluorescent dye is removed by size exclusion chromatography on a G-50 Sephadex column.
Oligonucleotide Align 1 x 10 ^-5 moles of the ODN for cutting the fluorescent dye from the nucleotide was then diluted in TE (TE is 0.01M Tris, pH 7.0, EDTA 5mM) ( 12, 3 -fold dilution). Add 25 μl of 0.01 M dithiothreitol (DTT) to a volume of 100 μl of ODN. DDT is not added to the same set of controls. The mixture is incubated at room temperature for 15 minutes. Fluorescence is measured on a black microtiter plate. The solution is removed from the culture tube (150 占 퐇) and placed in a black microtiter plate (manufacturer: Dynatek Laboratories, Chantilly, VA). Using a plate excitation wavelength of 495 nm and a monitored emission wavelength of 520 nm for fluorescein and a fluorescence scan using a excitation wavelength of 591 nm and a monitoring emission wavelength of 612 nm for Texas Red and an excitation wavelength of 570 nm for lysamine and a monitoring emission wavelength of 590 nm II fluorescence meter (manufacturer: Flow Laboratories, Mclean, VA).
Mall of fluorescent pigmentRFU not cutREF TruncatedRFU Glass 1.0 × 10 ⁵ M6.412001345 3.3 × 10 ⁶ M2.4451456 1.1 × 10 ⁶ M0.9135130 3.7 × 10 ⁷ M0.34448 1.2 × 10 ⁷ M0.1215.316.0 4.1 × 10 ⁷ M0.144.95.1 1.4 x 10 ⁸ M0.132.52.8 4.5 × 10 ⁹ M0.120.80.9
The data indicate that fluorescence increases about 200-fold when the fluorescent dye is cleaved from the ODN.
Example 4
Preparation of tacked M13 sequence primers and demonstration of tack cleavage
(5'-hexylamine-TGTAAAACGACGGCCAGT-3 ") (SEQ ID NO: 1) 5'-terminally terminated amine-linked oligonucleotides Cyanuric chloride and 10% n = methyl-pyrrolidone alkaline (preferably pH 8.3 to 8.5) buffer at 19 ° C to 25 ° C for 30 minutes to 120 minutes. The final reaction conditions are pH 8.3 0.15 M sodium borate, 2 mg / ml recrystallized cyanuric acid chloride and 500 g / ml individual oligonucleotides. Unreacted cyanuric acid chloride was removed by size exclusion chromatography on a G-50 Sephadex column do.
The active purified oligonucleotide is then reacted with 100 times molar excess cystamine in 0.15 M sodium borate at pH 8.3 for 1 hour at room temperature. The unreacted cystamine is removed by G-50 Sephadex size exclusion chromatography. The derived ODN is then reacted with various amides. The derived ODN preparation was divided into twelve pieces and each piece was divided into three parts: (1) 4-methoxybenzoic acid, (2) fluorobenzoic acid, (3) toluenic acid, (4) benzoic acid, Acetic acid, (6) 2,6-difluorobenzoic acid, (7) nicotinic acid N-oxide, (8) 2-nitrobenzoic acid, (9) 5-acetylsalicylic acid, Cinnamic acid, and (12) 3-aminonicotinic acid. The reaction is carried out at 37 [deg.] C for 2 hours with 0.2 M Na borate pH 8.3 (25 molar excess). The derived ODN is purified by gel exclusion chromatography on a G-50 Sephadex column.
Oligonucleotide Align 1 x 10 ^-5 moles of the ODN to cut the tack from the nucleotide diluted in TE (TE is 0.01M Tris, pH 7.0, 5mM EDTA) in EtOH (V / V) and 50% (12, 3-fold dilution ). Add 25 μl of 0.01 M dithiothreitol (DTT) to a volume of 100 μl of ODN. DDT is not added to the same set of controls. The mixture is incubated at room temperature for 30 minutes. 0.1 M NaCl is added and 2 volumes of EtOH are added to promote ODN. ODN is removed from the solution by centrifugation at 14000 x G for 15 minutes at < RTI ID = 0.0 > 4 C. < / RTI > The supernatant is preserved and completely dried. The pellet is dissolved in 25 [mu] l of MeOH. The pellet is then tested for the presence of tack by mass spectrometry.
The mass spectrometer of this work uses an ion source Fourier-transform mass spectrometer (FTMS). Samples prepared for MALDI analysis are directly deposited at the tip of the probe and inserted into the ion source. When the sample is irradiated with a laser wave, ions are extracted from the source, passed through a long, quadrupole ion guide that focuses, and moved to a FTMS analyzer cell located in the hole of the superconducting magnet.
The spectrum derives the following information. Peaks varying in intensity within the relative intensity units of 25-100 at the following molecular weights: (1) 212.1 amu (atomic mass unit) representing a 4-methoxybenzoic acid derivative. (3) toluene acid derivative showing 4-fluorobenzoic acid derivative, 196.1 amu showing (4) benzoic acid derivative, (5) 235.2 amu indicating (5) indole- Difluorobenzoic acid derivative, (7) 199.1 amu representing a nicotinic acid N-oxide derivative, (8) 227.1 amu representing a 2-nitrobenzamide acid derivative, (9) 5-acetylsalicylic acid (10) 226.1 amu representing a 4-ethoxybenzoic acid derivative, 209.1 amu representing (11) cinnamic acid derivative, (12) 198.1 amu representing a 3-aminonicotinic acid derivative.
The results indicate that the MW-stagnating agent can be cleaved from the primers and detected by mass spectrometry.
Example 5
Preparation of a compound of the formula R _1-36 -LYS (ε- _I NIP) -ANP-TFP
Figure 3 shows a parallel synthesis of a set of 36 TLX compounds (X = L _h ), wherein L _h is an active ester (especially tetrafluorophenyl ester), L ² is a methylene group linking L _h and L ² Nitrobenzylamine group having L ³ , which is L ^3, which has a modular structure to form an amide bond when the carboxylic acid group of the lysine bonds with the nitrogen atom of the L ² benzylamine group, and the variable weight component R _1-36 ( Where the R group corresponds to T < ² > as defined herein and can be introduced via any particular carboxylic acid as described herein, can be introduced into the mass spectrometric sensitivity enhancer group (introduced via N-methylisonipecotic acid Amino group of the lysine, while binding through the [alpha] -amino group of the lysine.
3
Step A
NovaSyn HMP resin (manufacturer: NovaBiochem; 1 equivalent) is suspended in a collection vessel of ACT357 with DMF. Compound I (ANP manufacturer: ACT; 3 eq.), HATU (3 eq.) And NMM (7.5 eq.) In DMF are added and the collection vessel is shaken for 1 h. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling and washing steps for resin I are repeated, yielding compound II.
Step B
The resin (Compound II) is mixed with 25% piperidine in DMF and shaken for 5 minutes. The resin is filtered and then mixed with 25% piperidine in DMF and shaken for 10 minutes. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X) and DMF (2X) and used directly in Step C.
Step C
The deprotected resin from step B was suspended in DMF and the FMOC-protected amino acid containing the protected amine function in DMF in the side chain (Fmoc-Lysine (Aloc) -OH, manufacturer: PerSeptive Biosystems; 3 equivalents), HATU (3 eq) and NMM (7.5 eq.). The container is shaken for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X), and DMF (2X). The coupling and washing steps of Fmoc-Lys (Aloc) -OH to the resin are repeated to give compound IV.
Step D
An aliquot of the resin (Compound IV) is washed with CH ₂ Cl ₂ (2X) and suspended in a solution of (PPh ₃ ) ₄ Pd (0) (0.3 eq) and PhSiH _{3 in} CH ₂ Cl ₂ . The mixture is shaken for 1 hour. The solvent is removed and the resin is washed with CH ₂ Cl ₂ (2X). The palladium step is repeated. The solvent is removed and the resin is washed with N, N-diisopropylethylammonium diethyldithiocarbamate, DMF (2X) in CH ₂ Cl ₂ (2X), DMF (2X) to give compound V.
Step E
The deprotected resin from Step D is coupled as described in Step C with N-methylisonipecotic acid to give compound VI.
Step F
Fmoc protected resin VI is equally distributed from the collection vessel to 36 reaction vessels by ACT 357 to give compounds VI _1-36 .
Step G
The resin (Compound VI _1-36 ) is treated with piperidine as described in Step B to remove the FMOC group.
Step H
36 aliquots of the deprotected resin from Step G are suspended in DMF. To each reaction vessel is added the appropriate carboxylic acid (R _1-36 CO ₂ H; 3 equivalents), HATU (3 equivalents) and NMM (7.5 equivalents) in DMF. The container is shaken for 1 hour. The solvent is removed and an aliquot of the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling and washing steps of R _1-36 CO ₂ H to aliquots of resin are repeated to give compounds VIII _1-36 .
Step I
An aliquot of the resin (Compound VIII _1-36 ) is washed with CH ₂ Cl ₂ (3X). The _{CH 2 Cl 2 90:: H20} : TFA to each reaction vessel 5: was added to 5, the vessel is agitated for 120 minutes. The solvent is filtered from the reaction vessel into individual tubes. An aliquot of the resin is washed with CH ₂ Cl ₂ ( ₂ ×) and MeOH (2 ×), and the filter is combined into individual tubes. The individual tubes are evaporated in vacuo to form compounds IX _1-36 .
Step J
Each free carboxylic acid IX _1-36 is dissolved in DMF. To each solution was added pyridine (1.05 eq.) And tetrafluorophenyltrifluoroacetate (1.1 eq.) Was added. The mixture is stirred for 45 minutes at room temperature. The solution is diluted with EtOAc, washed with 5% aqueous NaHCO ₃ (3X), dried over Na ₂ SO ₄ , filtered and evaporated in vacuo to form compounds X _1-36 .
Example 6
Preparation of a compound of the formula R _1-36 -LYS (ε- _I NIP) -NBA-TFP
4 is 36 TLX compounds (X = L _h) to indicate the parallel synthesis of a set, at this time, L _h is activated ester (specifically, tetrafluoroethylene-phenyl ester), L ² is L _h is an aromatic ring directly to the L ² group An ortho-nitrobenzylamine group having L ³ that is a direct bond of L _h and L ² when bonded, T is a modular structure that forms an amide bond when the carboxylic acid group of the lysine is bonded to the nitrogen atom of the L ² benzylamine group , And the variable weight components R _1-36 , where the R group corresponds to T ² as defined herein and can be introduced via any particular carboxylic acid described herein, can also be incorporated into the mass spectral sensitization enhancer group Amino acid) introduced through the -Amino group of the lysine, while binding through the -Amino group of the lysine.
See FIG.
Step A
The NovaSyn HMP resin was coupled with NBA prepared according to the procedure for Compound I (Brown et al., Molecular Diversity, 1, 4 (1995)) as described for Example 5, Step A Compound II is obtained.
Step B-J
The resin (Compound II) is treated as described in Example BJ step to give compounds X _1-36 .
Example 7
Preparation of a compound of formula _I NIP-LYS ( -R _1-36 ) -ANP-TFP
Figure 5 shows a parallel synthesis of a set of 36 TLX compounds (X = L _h ), wherein L _h is the active ester (especially tetrafluorophenyl ester), L ² is the methylene group linking L _h and L ² the L ³ is ortho with - nitrobenzyl amine group, T has a modular structure which forms an amide bond when the carboxylic acid group of lysine combined with the nitrogen atom of the L ² benzylamine group, a variable weight component R _1-36 ( Where the R group corresponds to T < ² > as defined herein and can be introduced via any particular carboxylic acid as described herein, can be introduced into a mass spectrometric sensitizer group (introduced via N-methylisonipecotic acid Amino group of the lysine, while binding through the -Amino group of lysine.
5
Step A-C.
Same as Example 5.
Step D
The resin (Compound IV) is treated with piperidine as described in Step B of Example 5 to remove the FMOC group.
Step E
Amine in Step D is coupled with N-methylisocyanic acid as described in Step D of Example 5 to give compound V. The reaction is carried out in the presence of a base.
Step F
Same as Example 5.
Step G
The resin (Compound VI _1-36 ) is treated with palladium as described in Step D of Example 5 to remove the Aloc group.
Step HJ
Compound X _1-36 is prepared in the same manner as in Example 5.
Example 8
Preparation of a set of compounds of the formula R _1-36 -GLU (γ-DIAEA) -ANP-TFP
Figure 6 shows a parallel synthesis of a set of 36 TLX compounds (X = L _h ) wherein L _h is an active ester (especially tetrafluorophenyl ester), L ² is a methylene group linking L _h and L ² ortho with L ³ being a-nitrobenzyl amine group, T has a modular structure which forms an amide bond when the α- carboxylic acid group of glutamic acid Mart combined with the nitrogen atom of the L ² benzylamine group, a variable weight component R _1-36 , where the R group corresponds to T ² as defined herein and can be introduced via any particular carboxylic acid as described herein, can also be a mass spectral sensitization enhancer group (2- (diisopropyl Diamino) ethylamine binds through the -Carboxylic acid of glutamic acid while it binds through the -Amino group of glutamic acid group.
6
Step A-B.
Same as Example 5.
Step C
The deprotected resin (Compound III) is coupled to Fmoc-Glu- (OAI) -OH using the coupling method described in Step C of Example 5 to give compound IV.
Step D
The allyl ester on resin (Compound IV) was washed with CH ₂ Cl ₂ (2X) and a solution of (PPh ₃ ) ₄ Pd (0) (0.3 eq) and N-methylaniline (3 eq) in CH ₂ Cl ₂ Mix. The mixture is shaken for 1 hour. The solvent is removed and the resin is washed with CH ₂ Cl ₂ (2X). The palladium step is repeated. The solvent is removed and the resin is washed with N, N-diisoporphylethylammonium diethyldithiocarbamate, DMF (2X) in CH ₂ Cl ₂ (2X), DMF (2X) to give compound V.
Step E
The deprotected resin from Step D is suspended in DMF and HATU (3 eq) and NMM (7.5 eq.) Are mixed and activated. The container is shaken for 15 minutes. The solvent is removed and the resin is washed with NMP (1X). The resin is mixed with 2- (diisopropylamino) ethylamine (3 eq) and NMM (7.5 eq). The container is shaken for 1 hour. The coupling and washing steps of 2- (diisopropylamino) ethylamine to the resin are repeated to give compound VI.
Step F-J
Same as Example 5.
Example 9
Preparation of a compound of the formula R _1-36 -LYS (ε-INIP) -ANP-LYS (ε-NH ₂ ) -NH ₂
Figure 7 shows a parallel synthesis of a set of 36 TLX compounds (X = L _h ), wherein L _h is the active ester (in particular the ε-amino group of the lysine-derived moiety), L ² is L _h and L ² Nitrobenzylamine group having L ³ , which is an alkylene aminoacylalkylene group substituted with carboxamido which binds to L ² benzylamine group, and T denotes an amido bond when the carboxylic acid group of lysine is bonded to the nitrogen atom of L ² benzylamine group And wherein the variable weight components R _1-36 , where the R group corresponds to T ² as defined herein and can be introduced via any particular carboxylic acid described herein, The spectral sensitization enhancer group (introduced via N-methylisonipecotic acid) binds through the [epsilon] -amino group of lysine, but through the [alpha] -amino group of lysine.
7
Step A
Fmoc-Lys (Boc) -SRAM resin (manufacturer: ACT; Compound I) is mixed with 25% piperidine in DMF and shaken for 5 minutes. The resin is filtered, mixed with 25% piperidine in DMF and shaken for 10 minutes. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X) and DMF (2X) and used directly in Step B.
Step B
The resin (Compound II), ANP (manufacturer: ACT; 3 equivalents), HATU (3 equivalents) and NMM (7.5 equivalents) in DMF are added and the collection vessel is shaken for 1 hour. The solvent is removed and the resin is washed with NMP (2X), MeOH (2X) and DMF (2X). The coupling and washing steps of I for the resin are repeated to give compound III.
Step C-J
The resin (Compound III) is treated as in Step B-I of Example 5 to give compound X _1-36 .
Example 10
Preparation of a compound of the formula R _1-36 -LYS (ε-TFA) -LYS (ε- _I INP) -ANP-TFP
Figure 8 shows a parallel synthesis of a set of 36 TLX compounds (X = L _h ), wherein L _h is the active ester (especially tetrafluorophenyl ester), L ² is the methylene group linking L _h and L ² the L ³ the ortho with which-nitrobenzyl amine group, T has a modular structure which forms an amide bond when the carboxylic acid group of the first lysine coupling with the nitrogen atom of the L ² benzylamine group, mass spectral sensitivity enhancers (N -Methylisonipecotic acid) binds through the [epsilon] -amino group of the first lysine when the second lysine derivative binds to the first lysine through the [alpha] -amino group of the first lysine , The molecular weight-adapting group (with the trifluoroacetyl structure) is linked via the epsilon-amino group of the second lysine and the variable weight component R _1-36 , wherein the R group is attached to T ² as defined herein correspondence When and if it can be introduced through any particular carboxylic acid as described herein) is bonded through the α- amino group of the second lysine.
8
Step A-E
These steps are the same as Step A-E of Example 5.
Step F
The resin (Compound VI) is treated with piperidine as described in Step B of Example 5 to remove the FMOC group.
Step G
The deprotected resin (Compound VII) is coupled to Fmoc-Lys (Tfa) -OH using the coupling method described in Step C of Example 5 to give compound VIII.
Step H-K
The resin (Compound VIII) is treated as in Example 5, Step FJ to give compound XI _1-36 .
Example 11
Preparation of a compound of the formula R _1-36 -LYS (ε- _I INP) -ANP-5'-AH-ODN
Figure 9 shows a parallel set of 36 TLX compound sets (X = MOI, where MOI is a nucleic acid fragment, ODN) derived from the ester of Example 5 (the same procedure as other TLX compounds can be used when X is the active ester) Lt; / RTI > The MOI is conjugated to the T-L via the phosphodiester-alkylphenamine group via 5 ' at the end of the MOI.
9
Step A
Compound XII _1-36 is prepared according to the modified biotinylation procedure provided in Van Ness st al., Nucleic Acids Res., 19, 3345 (1991). To a solution of one of the 5'-aminohexyl oligonucleotides (compounds XII _{1-36, 1} mg) in 200 mM sodium borate (pH 8.3, 250 mL) was added tetrafluorophenyl ester (compounds X _1-36 from Example A, 100-fold molar excess in 250 mL of NMP) is added. The reaction is incubated overnight at room temperature. The unreacted hydrolyzed tetrafluorophenyl ester is removed from the compound XII _1-36 by Sephadex G-50 chromatography.
Example 12
Preparation of a compound of the formula R _1-36 -LYS (ε- _I INP) -ANP-LYS (ε- (MCT-5'AH-ODN)) -NH ₂
10 shows a parallel synthesis of a set of 36 TLX compounds (X = MOI, where MOI is a nucleic acid fragment, ODN) derived from an amine of Example 11, wherein the same procedure as for other TLX compounds can be used when X is an amine . The MOI is conjugated to the T-L via the phosphodiester-alkylene amine group 5 ' at the end of the MOI.
10
Step A
5 '- [6- (4,6-dichloro-1,3,5-triazin-2-ylamino) hexyl] oligonucleotide XII _1-36 was prepared as described in Van Ness st al., Nucleic Acids Res. , 19, 3345 (1991).
Step B
(4,6-dichloro-1,3,5-triazin-2-ylamino) hexyl] oligonucleotide (Compound XII ₁ (SEQ ID NO: ₁ )) at a concentration of 1 mg / ml in 100 mM sodium borate _-36 ) was added to a solution of one of the primary amines selected from R _1-36 -Lys (e-iNIP) -ANP-Lys (e-NH ₂ ) -NH ₂ (Compound X _1-36 from Example 11) Add a molar excess of 100 times. The solution is mixed overnight at room temperature. Unreacted amines are removed from compounds XII _1-36 by ultrafiltration through a 3000 MW cut membrane using H ₂ O as a wash solution (manufacturer: Amicon, Beverly, Mass.). Compound XIII _1-36 is liberated by reducing the volume to 100 mL.
Example 13
Demonstration of multiple tandem detection by mass spectrometry
This example describes the ability to detect multiple compounds (tags) simultaneously by mass spectrometry. In this particular embodiment, 31 compounds are mixed with the matrix, precipitated, dried to solid support, and then desorbed with a laser. The ion product is then introduced into the mass spectrometer.
(121.14), nicotinamide (122.13), pyrazinamide (123.12), and the like were mixed in the same moles of the following compound (purchased from Aldrich, Milwaukee, Wis.) To give a final concentration per compound of 0.002M (127.10), 2-thiophenecarboxamide (127.17), 4-aminobenzamide (135.15), toluimide (135.17), 6- (137.14), nicotinamide N-oxide (138.12), 3-hydropicolinamide (138.13), 4-fluorobenzamide (139.13), cinnamamide (147.18), 4- methoxybenzamide 151.17), 2,6-difluorobenzamide (157.12), 4-amino-5-imidazole-carboxamide (162.58), 3,4-pyridine-dicarboxamide (165.16), 4- ethoxybenzamide Nitrobenzamide (166.14), 3-fluoro-4-methoxybenzoic acid (170.4), indole-3-acetamide (174.2), 5- Ace (189.19), 1-naphthaleneacetamide (185.23), 8-chloro-3,5-diamino-2-pyrazinecarboxamide (187.59), 4- Amino-5-phenyl-4-pyrazole-carboxamide (202.22), 1-methyl-2-benzyl- malonamate (207.33), 4- amino- 2,3,5,6-tetrafluorobenzamide (208.11), 2,3-naphthalenedicarboxylic acid (212.22). The compounds are placed in DMSO at the concentrations described above. One l of the material is then mixed with an alpha-cyano-4-hydroxycinnamic acid matrix (after dilution 1: 10000) and precipitated in solid stainless steel.
The material is then desorbed with a laser using a Protein TOF mass spectrometer (manufacturer: Bruker, Manning Park, MA) and the ions formed are measured in both linear and reflective modes. The following m / z values are observed.
(Fig. 11)
121.1 - > benzamide (121.14)
122.1 - > - Nicotinamide (122.13)
123.1 -> pyrazinamide (123.12)
124.1
125.2
127.3 - > 3-Amino-4-pyrazolecarboxylic acid (127.10)
127.2 - > 2-Thiophenecarboxamide (17.17)
135.1 - > 4-Aminobenzamide (135.15)
135.1 - > Toluide (135.17)
136.2 - > 6-Methylnicotinamide (136.15)
137.1 - > 3-Aminonicotinamide (137.14)
138.2 -> Nicotinamide N-oxide (138.12)
138.2 - > 3-Hydro picolinamide (138.13)
139.2 - > 4-Fluorobenzamide (139.13)
140.2
147.3 ----> Cinnamamide (147.18)
148.2
149.2
4-Methoxybenzamide (151.17)
152.2
2,6-difluorobenzamide (157.12)
158.3
4-Amino-5-imidazole-carboxamide (162.58)
163.3
165.2 - > 3,4-Pyridine-dicarboxamide (165.16)
165.2 - > 4-Ethoxybenzamide (165.19)
166.2 -> 2,3-Pyrazinecarboxamide (166.14)
166.2 - > 2-Nitrobenzamide (166.14)
3-Fluoro-4-methoxybenzoic acid (170.4)
171.1
172.2
173.4
Indole-3-acetamide (174.2)
178.3
179.3 - > 5-Acetylsalicylamide (179.18)
181.2 - > 3,5-Dimethoxybenzamide (181.19)
182.2 ---->
1-naphthaleneacetamide (185.23)
186.2
8-chloro-3,5-diamino-2-pyrazinecarboxamide (187.59)
188.2
189.2 - > 4-Trifluoromethyl-benzamide (189.00)
190.2
191.2
192.3
5-Amino-5-phenyl-4-pyrazole-carboxamide (202.22)
203.2
203.4
1-methyl-2-benzyl-malonamate (207.33)
4-Amino-2,3,5,6-tetrafluorobenzamide (208.11)
212.2 2,3 Naphthalene dicarboxylic acid (212.22)
219.3
221.2
228.2
234.2
237.4
241.4
The data represent 22 of the 31 mixtures in the spectrum with the predicted mass, and nine of the 31 mixtures exceed the predicted mass in the spectrum with n + H mass (1 amu). The latter phenomenon may be due to quantization of amines in the compound. Thus, 31 of 31 compounds are detected by MALDY mass spectrometry. More importantly, the embodiment illustrates that a plurality of taps can be simultaneously detected by a spectroscopic analysis method.
The alpha-cyano matrix alone provides peaks at 146.2, 164.1, 172.1, 173.1, 189.1, 190.1, 191.1, 192.1, 212.1, 224.1, 228.0, 234.3. Other stagnant identities in the spectrum are due to efforts to refine the contaminants in the purchased compounds.
Example 14 Microsatellite marker: PCR amplification
Microsatellite markers are amplified using the following standard PCR conditions. To summarize, the PCR reaction is carried out in a total volume of 50 占 퐇 and contains 40 ng of genomic DNA, 50 pmol of each primer, 0.125 mM dNTP and 1 unit of Taq polymerization element. The 1X amplification buffer will contain 10mM Tris base, pH 9, 50 mM KCI, 1.5 mM MgCl 2, 0.1% Triton X-100 and 0.01% gelatin. The reaction is carried out using the " thermal initiation " step: the Taq polymeric element is added only after the initial denaturation step for 5 minutes at 96 < 0 > C. Amplification is performed for 35 cycles: denaturation (40 seconds at 94 占 폚) and annealing (55 占 폚 for 30 seconds). The elongation step (72 ° C for 2 minutes) ends the process after the final anneal. The obtained amplification product is short (90 to 350 base pairs long) and the temperature rise time interval from 55 占 폚 to 94 占 폚 (obtained at a ramping rate of 1 占 폚 / sec) Can be obtained.
Although specific embodiments of the invention have been described herein for purposes of illustration, it is to be understood that various modifications may be made without departing from the spirit and scope of the invention.

权利要求:
Claims (48)
[1" claim-type="Currently amended] (a) forming a tactile nucleic acid molecule from one or more selected target nucleic acid molecules, wherein the tack is correlated with a specific nucleic acid fragment and can be detected by non-fluorescent spectroscopy or potentiometry;
(b) separating the set molecules by size;
(c) cutting the tack from the tacked molecule and
(d) detecting the tack by non-fluorescence spectrometry or potentiometry, and then determining the identity of the nucleic acid molecule therefrom.
[2" claim-type="Currently amended] (a) combining with a target nucleic acid molecule a nucleic acid probe tacked for a sufficient time and under conditions permitting hybridization of the tacked nucleic acid probe to the complementarily selected target nucleic acid sequence, wherein the tacked nucleic acid probe is non- Can be detected by spectroscopic analysis or by potentiometric methods);
(b) changing the size of the hybridized and tapped probe, the size of the non-hybridized probe or target molecule, or the size of the probe: target hybrid;
(c) separating the tacked probe by size;
(d) cutting the tack from the tacked probe; and
(e) detecting the tack by non-fluorescence spectroscopy or potentiometric methods, and then detecting the nucleic acid molecule selected therefrom.
[3" claim-type="Currently amended] The method according to claim 1 or 2, wherein the detection of the tack is performed by mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy or constant potential current method.
[4" claim-type="Currently amended] 6. The method of claim 1, wherein at least four nucleic acid fragments are generated, each tack being unique to the selected nucleic acid fragment.
[5" claim-type="Currently amended] 3. The method of claim 2 wherein at least four nucleic acid probes are used, each tack being unique to the selected nucleic acid probe.
[6" claim-type="Currently amended] 3. The method according to claim 1 or 2, wherein the target nucleic acid molecule is generated by primer extension.
[7" claim-type="Currently amended] 3. The method of claim 2, wherein the size of the hybridized and tapped probe, the size of the non-hybridized probe or target molecule, or the size of the probe: target hybrid is greater than the polymerase extension, ligation, exonucleolysis, and endonuclease Lt; RTI ID = 0.0 > hydrolysis < / RTI >
[8" claim-type="Currently amended] 3. The method according to claim 1 or 2, wherein the tapped molecules are separated by a method selected from the group consisting of gel electrophoresis, capillary electrophoresis, micro-channel electrophoresis, HPLC, size exclusion chromatography and filtration.
[9" claim-type="Currently amended] 3. The method according to claim 1 or 2, wherein the tapped molecule is selected from the group consisting of an oxidation method, a reduction method, an acid stable method, a base instability method, an enzyme method, an electrochemical method, a thermal method and a photo instability method How to cut.
[10" claim-type="Currently amended] 4. The method according to claim 1 or 3, wherein the tack is detected by flight time-of-flight mass spectrometry, quadrupole mass spectrometry, magnetic sector mass spectrometry and electrical sector mass spectrometry.
[11" claim-type="Currently amended] 11. The method of claim 10, wherein the tack is detected by a constant potential current method using a detector selected from the group consisting of an electrical detector and a current detector.
[12" claim-type="Currently amended] 3. The method of claim 1 or 2, wherein the separation, cutting and detection steps are performed in a continuous manner.
[13" claim-type="Currently amended] 3. The method of claim 1 or 2, wherein the separation, cutting and detection steps are performed in a continuous manner in a single apparatus.
[14" claim-type="Currently amended] 14. The method of claim 13, wherein the separating, cutting and detecting steps are automated.
[15" claim-type="Currently amended] 3. The method of claim 1 or 2, wherein the tacked molecule or probe is generated from a 5 ' -tagged oligonucleotide primer.
[16" claim-type="Currently amended] 3. The method of claim 1 or 2, wherein the tacked molecule or probe is generated from a tethered dideoxynucleotide terminator.
[17" claim-type="Currently amended] (a) generating a tacked nucleic acid molecule from a selected target molecule, wherein the tack correlates with a specific nucleic acid fragment and can be detected by non-fluorescent spectroscopy or potentiometry;
(b) separating the set molecule by the length of the sequence;
(c) cutting the tack from the tacked molecule and
(d) detecting a tack by non-fluorescence spectroscopy or potentiometry, and then measuring the genotype of the organism from the tack.
[18" claim-type="Currently amended] (a) blending a nucleic acid molecule with a tapped set of conditions and a time sufficient to permit hybridization of the tacked molecule to the target molecule with the selected target molecule, wherein the tack is correlated with the specific nucleic acid fragment, Or can be detected by a potential difference method);
(b) separating the set molecule by the length of the sequence;
(c) cutting the tack from the tacked molecule and
(d) detecting the tack by non-fluorescence spectroscopy or potentiometry, and then measuring the genotype of the organism from the tack.
[19" claim-type="Currently amended] 19. The method of claim 17 or 18, wherein the tacked molecule is generated from a mixture of clones selected from the group consisting of genomic clones, cDNA clones, and RNA clones.
[20" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the tapped molecule is produced by a polymerase chain reaction.
[21" claim-type="Currently amended] The method according to claim 17 or 18, wherein the detection of the tack is performed by mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy or constant potential current method.
[22" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein at least four nucleic acid molecules are generated, each tack being unique to the selected nucleic acid fragment.
[23" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the target nucleic acid molecule is generated by primer extension.
[24" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the tapped molecules are separated by a method selected from the group consisting of gel electrophoresis, capillary electrophoresis, micro-channel electrophoresis, HPLC, size exclusion chromatography and filtration.
[25" claim-type="Currently amended] 19. The method of claim 17 or 18, wherein the tapped molecule is selected from the group consisting of an oxidation method, a reduction method, an acid instability method, a base instability method, an enzymatic method, an electrochemical method, a thermal method and a photo instability method How to cut.
[26" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the tack is detected by time-of-flight mass spectrometry, quadrupole mass spectrometry, magnetic sector mass spectrometry and electrical sector mass spectrometry.
[27" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the tack is detected by a constant potential current method using a detector selected from the group consisting of an electrical detector and a current detector.
[28" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the separating, cutting and detecting steps are performed in a continuous manner.
[29" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the separation, cutting and detection steps are performed in a continuous manner in a single apparatus.
[30" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the separation, cutting and detection steps are automated.
[31" claim-type="Currently amended] 19. The method according to claim 17 or 18, wherein the tacked molecule is generated from an oligonucleotide primer set to a non-3'-tack.
[32" claim-type="Currently amended] 19. The method of claim 17 or 18, wherein the tapped molecule is generated from a tiled dideoxynucleotide terminator.
[33" claim-type="Currently amended] 18. The method according to any one of claims 1 to 17, wherein the target molecule is obtained from a biological sample.
[34" claim-type="Currently amended] A composition comprising a plurality of compounds of formula (15).
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any atom selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows a moiety containing T ^ms to be cleaved from the remaining compound, wherein the moiety containing T ^ms is mono-ionized when the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids Lt; / RTI > functional group that carries a charged state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI;
Two or more compounds have the same T ^ms , but the MOI groups of these molecules have non-identical nucleotide lengths.
[35" claim-type="Currently amended] A composition comprising a plurality of compounds of formula (15).
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any atom selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows a moiety containing T ^ms to be cleaved from the remaining compound, wherein the moiety containing T ^ms is mono-ionized when the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids Lt; / RTI > functional group that carries a charged state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI;
Two or more compounds have the same T ^ms , but these compounds have an unequal elution time by column chromatography.
[36" claim-type="Currently amended] A composition comprising a plurality of compounds of formula (15).
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any atom selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows a moiety containing T ^ms to be cleaved from the remaining compound, wherein the moiety containing T ^ms is mono-ionized when the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids Lt; / RTI > functional group that carries a charged state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI;
Two compounds with nucleotide lengths of the same MOI also do not have the same T ^ms .
[37" claim-type="Currently amended] 36. The composition of any one of claims 34 to 36, wherein the plurality is greater than two.
[38" claim-type="Currently amended] 36. A composition according to any one of claims 34 to 36, wherein the majority is greater than 4.
[39" claim-type="Currently amended] 37. The composition of any one of claims 34 to 36, wherein the nucleic acid fragment has a sequence complementary to the vector portion, wherein the fragment is capable of priming the polynucleotide synthesis.
[40" claim-type="Currently amended] 36. The composition of any one of claims 34-36, wherein the T ^ms group of the multiple members is different by 2 amu or more.
[41" claim-type="Currently amended] 37. The composition of any one of claims 34 to 36, wherein the T ^ms group of the multiple members is different by 4 amu or more.
[42" claim-type="Currently amended] A composition comprising a plurality of each set of compounds of formula (15).
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any atom selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows a moiety containing T ^ms to be cleaved from the remaining compound, wherein the moiety containing T ^ms is mono-ionized when the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids Lt; / RTI > functional group that carries a charged state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI;
The members in the first set of compounds have the same T ^ms group, and more than ten members within the first set, there have non-identical MOI groups having different members of the nucleotides of the MOI, wherein T ^ms group among the set of It is different than 2amu.
[43" claim-type="Currently amended] 43. The composition of claim 42, wherein the plurality is at least 3.
[44" claim-type="Currently amended] 43. The composition of claim 42, wherein the plurality is at least 5.
[45" claim-type="Currently amended] A composition comprising a plurality of each set of compounds of formula (15).
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any atom selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows a moiety containing T ^ms to be cleaved from the remaining compound, wherein the moiety containing T ^ms is mono-ionized when the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids Lt; / RTI > functional group that carries a charged state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI;
The compounds in the set have the same elution time but the same T ^ms group.
[46" claim-type="Currently amended] Wherein at least one primer is a compound of formula (15), wherein each pair of primers is related to a different position.
Formula 15
T ^ms -L-MOI
In this formula,
T ^ms is an organic group detectable by mass spectroscopy, comprising carbon, at least one hydrogen and a fluoride, and any atom selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group that allows a moiety containing T ^ms to be cleaved from the remaining compound, wherein the moiety containing T ^ms is mono-ionized when the compound is mass spectroscopically analyzed and selected from tertiary amines, quaternary amines and organic acids Lt; / RTI > functional group that carries a charged state;
The MOI is a nucleic acid fragment, wherein L is conjugated to the MOI at a position other than the 3 'end of the MOI.
[47" claim-type="Currently amended] 47. The kit of claim 46, wherein the plurality is greater than or equal to three.
[48" claim-type="Currently amended] 47. The kit of claim 46, wherein the plurality is greater than or equal to five.

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同族专利:

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公开号 | 公开日

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JP2008200040A|2008-09-04|

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EP0962537A2|1999-12-08|

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EP0962537B1|2009-06-17|

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WO1997027325A2|1997-07-31|

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NZ331043A|1999-01-28|

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CZ228598A3|1998-12-16|

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AU2244897A|1997-08-20|

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WO1997027325A3|1998-04-02|

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CA2243546A1|1997-07-31|

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GR3033887T3|2000-11-30|

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CN1202260C|2005-05-18|

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EP0840804B1|2000-04-05|

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DE69739465D1|2009-07-30|

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CA2243546C|2008-04-15|

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EP0962537A3|2004-02-11|

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AU717945B2|2000-04-06|

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PL328239A1|1999-01-18|

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AT434054T|2009-07-15|

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AT191515T|2000-04-15|

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PT840804E|2000-10-31|

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DK0840804T3|2000-07-17|

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HU9901856A3|2001-10-29|

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KR100482915B1|2005-09-12|

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HU9901856A2|1999-09-28|

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ES2145580T3|2000-07-01|

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DE69701612T2|2000-08-31|

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CN1212020A|1999-03-24|

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DE69701612D1|2000-05-11|

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JP2000503845A|2000-04-04|

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BR9707016A|2000-01-04|

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EP0840804A1|1998-05-13|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1996-01-23|Priority to US1453696P

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1996-01-23|Priority to US60/014,536

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1996-06-04|Priority to US2048796P

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1996-06-04|Priority to US60/020,487

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1997-01-23|Application filed by 니아리 린다 제이, 라피진, 인코포레이티드

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1999-11-15|Publication of KR19990081924A

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2005-09-12|Application granted

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2005-09-12|Publication of KR100482915B1

优先权:

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申请号 | 申请日 | 专利标题

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US1453696P| true| 1996-01-23|1996-01-23|

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US60/014,536|1996-01-23|

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US2048796P| true| 1996-06-04|1996-06-04|

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US60/020,487|1996-06-04|