Methylation status detection procedures and kits

专利摘要:

公开号:ES2669214T9
申请号:ES12858081T
申请日:2012-12-13
公开日:2019-07-05
发明作者:John Arne Dahl；Adam Brian Robertson；Arne Klungland；Linda Ellevog
申请人:Oslo Universitetssykehus hf；
IPC主号:

专利说明:

[0001]
[0002] Mutilation status detection procedures and kits
[0003]
[0004] Field of the Invention
[0005]
[0006] The present invention relates to methods and kits for detecting 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5meC) in nucleic acid (eg, DNA, RNA). In some embodiments, the present invention relates to the detection of 5hmC in genomic DNA, eg, mammalian genomic DNA.
[0007]
[0008] Background of the invention
[0009]
[0010] The 5-hydroxymethylcytosine (5hmC) modification in mammalian DNA was discovered 30 years ago1. At that time, it was noted that the 5hmC modification was a rare and non-mutagenic DNA damage lesion2 and, therefore, little attention was given to it. In early 2009, 5hmC was identified again; However, this time the importance of 5hmC in epigenetics was understood, since two independent teams began the initial characterization of the 5hmC modification. A team identified an enzyme capable of catalyzing the formation of 5hmC from 5-methylcytosine - Tet13. The other team demonstrated that 5hmC was a stable modification present in specialized Purkinje neurons4. Further investigation has shown that Tet1, Tet2, and Tet3 are capable of catalyzing the oxidation of 5meC creating 5hmC5'7.
[0011]
[0012] The molecular function of 5hmC is not yet well understood; however, 5hmC has been shown to be related to various DNA transactions; It has been shown to be an intermediate product in the demethylation of DNA38, which has a dual function in transcription9'11 and, in the case of aberrant 5hmC patterns, which is related to tumorigenesis7. Although the function of the 5hmC modification is not yet clear, it has been clarified that the identification of genomic regions containing 5hmC can help elucidate the function of this base. This need to identify genomic regions that contain 5hmC has led to the development of appropriate procedures. Currently, several procedures are available to identify 5hmC; Each of these procedures has certain limitations, which are explained below. The procedure described herein allows a specific base resolution of (i) 5hmC and (ii) 5meC in DNA.
[0013]
[0014] Currently, there are several procedures that allow the identification of 5hmC. Such procedures include antibodies developed against 5hmC92122, antibodies developed against cytosine 5-methylene sulfonano (CMS) the product of 5hmC treatment with bisulfite723, real-time sequencing of single molecule based on the kinetics of DNA polymerase24, restriction enzymes that are resistant or sensitive at 5hmC or p-glu-5hmC25'27 and three procedures that take advantage of p-glucosyltransferase: (i) incorporation of a chemical marker in the substrate for p-gt28, (ii) glycosylation procedure, periodate oxidation and biotinylation (GLIB ) 23 and (iii) JPB1 immunoprecipitation assay targeting glu-5hmC12.
[0015]
[0016] The use of antibodies seems a reasonable option to identify DNA modifications; however, both the authors of the invention and other researchers5 have observed that part of the antibodies currently available directed against 5hmC seem to lack the ability to sufficiently enrich the DNA containing 5hmC; In fact, a report shows that a particular antiserum developed against 5hmC lacks the ability to differentiate 5hmC from 5meC5. It has been reported that antisera developed against 5hmC usually present a preference for dense genomic regions in 5hmC22 content. In addition, the use of polyclonal antisera directed against 5hmC is an inherent problem, since there will be a variation from one animal to another in the antigenic specificity for 5hmC that will affect the long-term usefulness of these antisera.
[0017]
[0018] After treatment with sodium bisulfite, 5hmC is converted to CMS, which, after sequencing, appears identical to 5meC converted with bisulfite; therefore, it has been shown that the use of bisulfite sequencing cannot distinguish between 5meC and 5hmC30. It should be noted that a team has developed an antiserum directed against CMS723.
[0019] Real-time sequencing of a single molecule (SMRT) takes advantage of the original Sanger sequencing technique; However, this procedure has the capacity to distinguish between cytosine, 5meC and 5hmC using the kinetic signature or speed with which the polymerase passes on each base24. This procedure, apart from being prohibitively expensive, requires a significant amount of DNA that is already enriched from 5hmC before use, which makes it dependent on the 5hmC enrichment assay. Since such a procedure employs high performance sequencing, it is complicated to analyze a single locus or a few loci.
[0020]
[0021] Several research teams and companies have identified restriction enzymes that are sensitive or resistant to 5hmC or p-glu-5hmC25-27. The principle behind these systems is that, after treatment with restriction enzymes, unmodified DNA is cleaved, with the result of a signal reduction in a qPCR reaction. Said signal reduction is then compared with an undigested sample and the difference in qPCR signals is proportional to the amount of 5hmC present in the initial sample. These procedures work perfectly for genomic regions that contain significant amounts of 5hmC; however, since the restriction sites recognized by these enzymes are 4-6 bp in length, these restriction endonuclease-based procedures, at best, can only recognize 1/16 of all modifications of 5hmC. US Patent 2006/00257 discloses a method for copying genomic DNA methylation patterns (MGD) during isothermal amplification of MGD. Said method comprises obtaining MGD, copying the methylation patterns of MGD using a DNA methylation maintenance enzyme, while MGD isothermally amplifying using DNA polymerase with chain displacement activity under conditions that simultaneously promote the activity of the DNA methylation enzyme and DNA polymerase with chain displacement activity and, optionally, the purification of MGD, as necessary to allow further manipulation.
[0022]
[0023] Three teams have developed procedures that take advantage of the specificity that p-gt has for 5hmC. The first team28 incorporated an azide group into the substrate for p-gt - UDP-glucose - creating UDP-6-N3-Glucose. Once the azide-modified glucose was incorporated into DNA containing 5hmC by p-gt, it was possible to add a second group to 6-N3-glu-5hmC using "click" chemistry. This second chemical group could contain a biotin for immunoprecipitation, a fluorescent probe for quantification and, theoretically, any group that could be coupled to the modified glucose using "click" chemistry. The main drawback of this procedure is that UDp-6-N3-glucose is not produced commercially and requires significant experience in organic chemistry for its synthesis. On the other hand, this directed 5hmC strategy has been combined with a primer extension assay and has been shown to allow a specific base resolution since a chemical group can bind with 6-N3-glu-5hmC that bites a DNA polymerase . By blocking the polymerase, it can be assumed that the terminal base originally contained a 5hmC modification. The use of this specific basic resolution procedure presents substantial problems, since it must be assumed that all the ends ending in C must be 5hmC. While it is possible to potentially average this effect with several high-performance sequencing readings assuming highly optimized enzyme-DNA relationships, it remains problematic for a single gene analysis.
[0024]
[0025] A second approach in which p-gt is used to identify genomic regions employs the glycosylation, periodate oxidation, biotinylation (GLIB) procedure 23 In this procedure, after transferring glucose to 5hmC the resulting p-glu-5hmC is oxidized using NaIO4 which creates reactive aldehydes in the glucose fraction bound to 5hmC. It is then possible to react these oxidized glucose molecules with aldehyde reactive probes containing commercially available biotin modification. Said biotinylation allows efficient immunoprecipitation of DNA containing 5hmC.
[0026]
[0027] Finally, the third approach in which p-gt is used for the identification of 5hmC involves the specific recognition of this modified base with a second protein-binding protein based on J or JPB1. Since the only difference between p-glucosyl-5hmC and base J is an amino group, it was deduced that JPB1 might be able to interact specifically with p-glu-5hmC. In fact, JPB1 was able to interact specifically with p-glu-5hmC12. Therefore, when JPB1 was covalently bonded with epoxy modified magnetic beads, immunoprecipitation of DNA containing p-glu-5hmC was possible. Once the immunoprecipitated DNA protein was extracted, it was demonstrated by gene-specific qPCR that it was possible to enrich the DNA containing 5hmC12. Mechanically, this procedure provides two degrees of specificity for the identification of 5hmC in genomic DNA: first, p-gt can only modify cytosines in the DNA that are hydroxymethylated and, secondly, JPB1 interacts specifically with p-glu-5hmC . Like all DNA immunoprecipitation procedures, the optimal resolution to a large extent of this procedure can be used to identify a 5hmC base within approximately 50-100 base pairs; This limitation is due to the inability to reliably identify DNA fragments of a shorter length by applying currently available molecular biology procedures. Another consideration when using this protocol is that this procedure may over-represent regions of DNA that contain high levels of 5hmC. Such possible over-representation could possibly take place because in the dense regions of 5hmC more JPB1 can interact with DNA and immunoprecipitate these regions more efficiently.
[0028]
[0029] Improved procedures are needed to detect 5-hydroxymethylcytosine residues in DNA. In particular, procedures that can discriminate between 5meC and 5hmC are needed, as well as procedures that can identify 5meC and 5hmC in single-base resolution.
[0030]
[0031] Summary of the invention
[0032]
[0033] The present invention relates to methods and kits for detecting 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5meC) in nucleic acid (eg, DNA, RNA). In some embodiments, the present invention relates to the detection of 5hmC in genomic DNA, eg, mammalian genomic DNA.
[0034]
[0035] In some embodiments, the present invention provides methods for detecting 5-methylated and hydroxymethylated cytosine residues in a nucleic acid sample comprising: replication of said nucleic acid sample under conditions such that said 5-methylated cytosine residues are maintained and dilute said hydroxymethylated cytosine residues; treatment of said replicated nucleic acid sample to convert the unmodified cytosine residues into uracil or thymidine residues; wherein the nucleic acid sample is divided into at least a first and a second portion and said replication and treatment steps are performed on said first portion, and comparison of the sequence of said first nucleic acid sequence with the sequence of said second nucleic acid portion, wherein said hydroxymethylated cytosine moieties are identified as moieties that are read by sequencing as a uracil or thymidine moiety in said first nucleic acid moiety and as a cytosine moiety in the corresponding position in said second nucleic acid portion and wherein the 5-methylated cytosine moieties are identified as moieties that are read as cytosine moieties in both first and second nucleic acid moieties. In some embodiments, replication of said first portion further comprises: a) replication of said nucleic acid with a labeled primer to provide a labeled replicated nucleic acid; b) treatment of said replicated nucleic acid chains labeled with a methyl transferase DNA to provide modified nucleic acid modified with labeled 5-methylcytosine; c) isolating said modified nucleic acid modified with labeled 5-methylcytosine; d) treatment of said replicated nucleic acid modified with bisulfite-labeled labeled 5-methylcytosine to convert unmodified cytosine residues into uracil residues; and e) replication of said labeled bisulfite treated nucleic acid isolated with a polymerase to provide a first portion of bisulfite treated nucleic acid. In some embodiments, the labeled primer is a biotinylated primer. In some embodiments, the other modified cytosine residues are selected from the group consisting of 5-hydroxymethyl cytosine and beta-glu-5-hydroxymethyl cytosine. Other examples of modified cytosine residues are alpha-glucosyl-5-hydroxymethylcytosine, beta-glucopyranosyl-alpha-glycosyl-5-hydroxymethylcytosine (gentiobiosyl-5-hydroxymethylcytosine), 5-formylcytosine and 5-carboxycytosine.
[0036]
[0037] In some embodiments, the replication of said first portion under conditions such that the 5-methylated cytosine residues are maintained and the 5-hydroxymethylated cytosine residues are diluted comprises the replication of said nucleic acid with a polymerase to provide replicated nucleic acid and the treatment of said nucleic acid replicated with an enzyme to give 5-methylated cytosine residues. In some embodiments, the steps of replication and treatment with an enzyme are performed one or more times. In some embodiments, the steps of replication and treatment with an enzyme are repeated 5 times or more. In some embodiments, the steps of replication and treatment with an enzyme are repeated 7 times or more. In some embodiments, the steps of replication and treatment with an enzyme are repeated 10 times or more. In some embodiments, the steps of replication and treatment with an enzyme are performed from about 1 to about 20 times or more. In some embodiments, replication is by polymerase chain reaction. In some embodiments, replication is by primer extension reaction. In some embodiments, the enzyme is a methyl transferase DNA. In some embodiments, the methyl transferase DNA is DNMT1. In some embodiments, the methyl transferase DNA is M.Sssl.
[0038]
[0039] In some embodiments, the treatment of said first and second portions to convert unmodified cytosine residues into thymidine residues further comprises treating said portions of first and second nucleic acid with bisulfite to convert unmodified cytosine residues into uracil residues and the replication of said first and second nucleic acid portions with a polymerase to convert said uracil residues to thymidine residues. In some embodiments, replication is performed 1 or more times. In some embodiments, replication is performed 5 times or more. In some embodiments, replication is performed 7 times or more. In some embodiments, replication is performed 10 times or more. In some embodiments, replication is repeated from about 1 to about 20 times. In some embodiments, replication is by polymerase chain reaction. In some embodiments, replication is by primer extension reaction.
[0040]
[0041] In some embodiments, the nucleic acid sample is selected from the group consisting of a human, plant, mouse, rabbit, hamster, primate, fish, bird, cow, sheep, pig, viral, bacterial and fungal nucleic acid sample .
[0042]
[0043] In some embodiments, the methods further comprise comparing the presence of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid of said sample with respect to a reference level, in which an increase or decrease in the level of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid is indicative of the presence of a disease or of the probable course of a disease. In some embodiments, the methods further comprise the step of providing a diagnosis or prognosis based on the increase or decrease of the level of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid compared to a reference level. In some embodiments, the disease is cancer. In some embodiments, the nucleic acid sample is genomic DNA.
[0044]
[0045] In some embodiments, the present invention provides methods for detecting methylated and hydroxymethylated cytosine residues in a nucleic acid sample comprising: a) dividing said sample into at least a first and second untreated portion; b) replication of said first portion with a labeled primer and a polymerase to provide labeled parental and replicated nucleic acid; c) treating said parental and replicated nucleic acid chains labeled with a methyl transferase DNA to provide modified nucleic acid modified with labeled 5-methylcytosine; d) isolating said modified nucleic acid modified with labeled 5-methylcytosine; e) treating said modified nucleic acid with bisulfite-isolated labeled 5-methylcytosine to convert unmodified cytosine residues into uracil residues; f) replication of said labeled bisulfite treated nucleic acid isolated with a polymerase to provide a first portion of bisulfite treated nucleic acid; g) sequencing said first portion of bisulfite treated nucleic acid; h) treating said second portion of nucleic acid with bisulfite to convert unmodified cytosine residues into uracil residues; i) replicating said bisulfite treated nucleic acid with a polymerase to provide a second portion of bisulfite treated nucleic acid; j) sequencing said second portion of bisulfite treated nucleic acid; and k) comparison of the sequence of said first bisulfite treated nucleic acid with the sequence of said second bisulfite treated portion, wherein the 5-hydroxymethylated cytosine residues are identified as residues that are read by sequencing as a residue of uracil or thymidine in said first portion of nucleic acid treated with bisulfite and as a cytosine residue in the corresponding position in said second portion of bisulfite treated nucleic acid and in which the residues of 5-methylated cytosine are identified as residues that are read as cytosine residues in said portions treated with first and second bisulfite. In some embodiments, said second portion is replicated with a polymerase before said sequencing step. In some embodiments, said steps b, c and d are repeated from about 2 to about 20 times. In some embodiments, said steps e and h are repeated from about 2 to about 20 times. In some embodiments, said replication of steps b, e and h is by polymerase chain reaction. In some embodiments, the methods further comprise comparing the presence of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid of said sample with a reference level, in which the increase or decrease of the level of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid is indicative of the presence of a disease or of the probable course of a disease. In some embodiments, the methods further comprise the step of providing a diagnosis or prognosis based on the increase or decrease of the level of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid compared to a reference level. In some embodiments, the disease is cancer. In some embodiments, the nucleic acid sample is genomic DNA.
[0046]
[0047] In some embodiments, the present invention provides a method for predicting a predisposition to a disease in a subject, diagnosing a disease in a subject, predicting the likelihood of recurrence of a disease in a subject, providing a prognosis for a subject with a disease , or selecting a subject with a disease for treatment with a particular therapy, which comprises: a) providing a genomic DNA sample of said subject; and b) detecting the methylation status of predetermined portions of said genomic DNA sample through the procedures described,
[0048] wherein an alteration of the methylation level of 5-hydroxymethylcytosine and / or 5-methylcytosine of said predetermined portions of said genomic DNA with respect to a state of reference methylation provides a selected indication of the group consisting of an indication of predisposition of the subject to a disease, an indication that the subject has a disease, an indication of the likelihood of recurrence of a disease in the subject, an indication of the subject's survival and an indication that the subject is a candidate for treatment with a particular therapy . In some embodiments, the disease is a cancer. In some embodiments, the subject is a human being.
[0049]
[0050] In some embodiments, the present invention provides a kit for determining the methylation status of a nucleic acid sample comprising: 1) container (s) with reagents for methylation of nucleic acid; and 2) container (s) with reagents for bisulfite sequencing and 3) a computer support comprising a computer program that analyzes the sequence data obtained with the kit according to the methods of the invention. In some embodiments, the kits further comprise nucleic acid primers for amplification and / or sequencing of a region of said nucleic acid sample.
[0051]
[0052] Other embodiments will become apparent to persons skilled in the corresponding subject on the basis of the instructions contained herein.
[0053]
[0054] Description of the drawings
[0055]
[0056] Figure 1. Schematic representation of certain embodiments of the present invention, in which bisulfite conversion and sequencing of untreated DNA "A" is applied which will be used as a reference to detect the total of 5meC and 5hmC. The procedure involves a 5hmC dilution test, the 5hmC dilution in the total cluster of DNA fragments while maintaining 5meC. Said dilution is achieved through sequential rounds of a PCR amplification cycle (dilution) and treatment of DNA with DNMT1 maintenance methyl transferase DNA that maintains enzymatic and specifically 5meC by adding a methyl group only to the unmethylated chain of products of hemimethylated PCR (in Figure 1 this sample is referred to as "B"). After a few rounds of this test, bisulfite conversion and sequencing of the treated DNA sample, B was applied. The bases that are read as cytosine of this sample must be protected against bisulfite conversion by 5meC and not by 5hmC . By comparing "B" with the reference sample "A" it is possible to easily detect all base positions containing 5hmC.
[0057]
[0058] Figure 2. The conversion of DNA with bisulfite results in the conversion of unmodified cytosine (C) into uracil (U) that will be read as thymine (T) after sequencing of the DNA amplified by PCR. Both 5meC and 5hmC are protected against conversion and will not be converted to U. Therefore, both bases will be read as C after sequencing. Bisulfite conversion is a well-established technology that has long been considered the benchmark for the detection of 5meC and only recently (2010) has it been reported in the scientific literature that bisulfite conversion cannot distinguish between 5meC and 5hmC.
[0059] Figure 3. Mouse DNMT1, human DNMT1 and M. SssI preferably hemi-5meC DNA methylate. 100 ng of each DNA substrate was incubated with 2 units of mouse DNMT1, human DNMT1 or Sssl methyl transferase as described in the heading "materials and procedures".
[0060]
[0061] Figure 4. Validation of the viability of the 5hmC dilution test. (A) The double stranded DNA oligo used in validation contains three CpG sites in which one is hemi-5meC, a second has no modification and a third is hemi-5hmC. (B) The conversion and sequencing with bisulfite of the lower unmodified chain of oligo In (A), when the oligo has not been treated with DNMT1, it showed that all C had been converted and read as T. (100% of T is equal to 16 of the 16 individual clones that are read as T in position C of the CpG site). (C) Treatment with DNMT1 before conversion and sequencing with bisulfite resulted in the addition of a methylated methyl group to C of the hemi-5meC CpG site in 87.5% of the oligos. (Sequencing gave a reading of a C at position C of the CpG site in 14 of the 16 clones). The addition of a methyl group was not observed transversely from C or 5hmC.
[0062]
[0063] Figure 5. Schematic representation of the procedure for a differential identification of 5hmC and 5meC in the specific basic resolution. (A) A follow-up scheme of the C bases of the CpG sites of a cDNA oligo that contains three CpG sites in which one has 5meC in both chains, a second has no modification and a third has 5hmC in both chains. CpG sites are monitored through a round of PCR (fusion, primer hybridization and elongation) and treatment with DNMT1 before the visualization of bisulfite and PCR treatment (30 cycles) that generates the bases that will be read in sequencing (B) Flowchart of the experimental procedure involved in the 5hmC dilution test.
[0064]
[0065] Figure 6. Preferential maintenance of 5meC with respect to 5hmC. The double stranded DNA oligo used in this case contains three CpG sites, one that has 5meC in both chains, a second that has no modification and a third that has 5hmC in both chains. (A) Conversion and sequencing with untreated oligo bisulfite showed that only modified Cs were protected against conversion (100% for both 5meC and 5hmC), while unmodified cytosines were all converted. (B) Taking the oligo of double stranded DNA through the three rounds of the dilution test, which involved PCR and treatment with DNMT1, before conversion and sequencing with bisulfite, the result was a preferential maintenance of 5meC with respect to 5hmC. There was no transverse methylation from 5hmC in any of the three rounds since the chains modified with initial 5hmC made up only 9% of the total grouping after the three rounds of the dilution test. (One would expect 50% after one round, 25% after two rounds and 12.5% after three rounds when there is no maintenance at all). The 5meC base was preferably maintained, with the result of a higher number of C protected in bisulfite conversion and a significantly higher reading than the 5hmC base. No addition of methyl group was observed transversely either from C or from 5hmC.
[0066]
[0067] Figure 7. Schematic representation of the differential identification procedure of 5hmC and 5meC in the specific base resolution with the use of the specific chain evaluation. (A) A follow-up scheme of the C bases of the CpG sites of a cDNA oligo containing three CpG sites, in which one has 5meC in both chains, a second has no modification and a third has 5hmC in both chains. The CpG sites are monitored in a round of PCR of specific chain primer extension (melt, primer hybridization and elongation) and treatment with DNMT1. The primer used may contain biotin label, or other label, to allow selection / isolation of the newly synthesized chain. The newly synthesized chain goes through treatment with bisulfite and PCR (30 cycles or other number) that generates the bases that will be read in the sequencing. (B) Flowchart of the experimental procedure in relation to the 5hmC dilution / loss test applying primer extension and specific chain evaluation.
[0068]
[0069] Figure 8. Amino acid sequence for DNMT1 (Mus musculus) Recombinant. Access number: GenBank: AAH53047.1 (SEQ ID NO: 1).
[0070]
[0071] Figure 9. Amino acid sequence for DNMT1 (Homo sapiens) Access number: GenBank: AAI44094.1 (SEQ ID NO: 2).
[0072]
[0073] Figure 10. Amino acid sequence for M.Sssl (Spiroplasma sp. (Strain MQ1)) site-specific methyl transferase DNA (SEQ ID NO: 3).
[0074]
[0075] Figure 11. Schematic representation of a 5hmC loss assay of the present invention using biotinylated primers and streptavidin capture beads. The upper right panel shows the results of the sequencing representative of 10 clones for the conventional bisulfite assay, with reference A, in which both 5meC and 5hmC are read as cytosine after treatment and the results of the sequencing of 10 clones for the methyl transfer assay / 5hmC loss test, with reference B, in which only 5meC will be read as cytosine after treatment. Cytosines in a CG sequence context (CpG) protected from bisulfite conversion are illustrated as black circles, while cytosines in a CG sequence context undergoing uracil deamination in bisulfite treatment are illustrated with blank circles. The combination of the reference bisulfite test data, A, in which both 5meC and 5hmC are read as cytosine after treatment and the methyl transfer test, B, in which 5meC is read as cytosine after treatment allows determine the position and quantity of 5hmC, from a simple calculation: AB = 5hmC. This quantification is described in the lower part of the right panel. These experimental results have been reproduced in 15 independent experiments.
[0076]
[0077] Figure 12. Scheme and graph in which the identification of two 5hmC containing CpG islands, which is 5hmC in a CG sequence, is presented in the human brain DNA TRIM31 gene applying the assay depicted in Figure 11. The positions of CpGs are represented schematically (not to scale) and the amount of 5hmC and 5meC in those positions for cytosine are given in the bar graph.
[0078] Figure 13. Bar graph showing the results of an experiment in which methyl transferase is blocked by the addition of a chemical group at 5hmC.
[0079]
[0080] Definitions
[0081]
[0082] In order to facilitate the understanding of the present invention, a series of terms and expressions are defined below:
[0083] As used herein, the term "sensitivity" is defined as a statistical measurement of the performance of an assay (eg, procedure, analysis), which is calculated by dividing the number of true positives by the sum of the true positives and false negatives.
[0084]
[0085] As used herein, the term "specificity" is defined as a statistical measurement of the performance of an assay (eg, procedure, analysis), which is calculated by dividing the number of true negatives by the sum of true Negative and false positive.
[0086]
[0087] As used herein, the term "informational" or the term "informational capacity" refers to a quality of a marker or panel of markers and, specifically, to the probability of finding a marker (eg, epigenetic marker; e.g., 5hmC, in one or more locations in particular) in a positive sample.
[0088]
[0089] As used herein, the term "dilution" refers to the reduction of non-5-methyl modified cytosine residues (eg, 5-hydroxymethyl cytosine residues) in a nucleic acid sample compared to 5-methyl cytosine residues through repeated rounds of replication of said DNA sample.
[0090]
[0091] As used herein, the term "modified cytosine residues not 5-methyl cytosine" refers to modified cytosine residues other than 5-methyl cytosine, for example, 5-hydroxymethyl cytosine, b-glu-5-hydroxymethyl cytosine, 5-formyl cytosine and 5-carboxycytosine.
[0092]
[0093] As used herein, the term "CpG Island" refers to a region of genomic DNA that contains a high percentage of CpG sites in relation to the incidence of average genomic CpG (for the same species, for the same individual or by subpopulation (eg, strain, ethnic subpopulation or the like.) There are several parameters and definitions for CpG islands; for example, in some embodiments, CpG islands are defined as having a GC percentage above 50% and with an observed / expected CpG ratio above 60% (Gardiner-Garden et al. (1987) J Mol. Biol. 196: 261-282; Baylin et al. (2006) Nat. Rev. Cancer 6: 107-116; Irizarry et al. (2009) Nat. Genetics 41: 178-186; which are incorporated by reference in their entirety.) In some embodiments, the CpG islands may have a GC content> 55% and an observed CpG / expected CpG ratio. 0.65 (Takai et al. (2007) PNAS 99: 3740-3745) There are also several parameters regarding the to length of the CpG islands. As used herein, CpG islands may be less than 100 bp; 100-200 bp, 200-300 bp, 300-500 bp, 500-750 bp; 750-1000 bp; 1000 or more bp in length. In some embodiments, the CpG islands exhibit methylation alteration patterns (eg, 5hmC alteration patterns) in relation to controls (eg, 5hmC methylation alteration in subjects with cancer with respect to subjects without cancer; tissue specific 5hmC alteration patterns; 5hmC alteration patterns in biological samples of subjects with neoplasia or tumor with respect to subjects without neuroplasia or tumor In some embodiments, the alteration of methylation implies an increase in the incidence of 5hmC In some embodiments, the alteration of the methylation implies a decrease in the incidence of 5hmC.
[0094]
[0095] As used herein, the terms "CpG shore" or "CpG Island shore" refer to a genomic region external to a CpG Island that has or has the potential to have methylation alteration patterns (eg. , 5hmC) (see, eg, Irizarry et al. (2009) Nat. Genetics 41: 178-186). The shores of CpG Island may also show patterns of alteration (e.g., 5hmC) with respect to controls (e.g., 5hmC alteration in subjects with cancer with respect to subjects without cancer; patterns of alteration of 5hmC tissue-specific; alteration of 5hmC in biological samples of subjects with neoplasia or tumor with respect to subjects without neoplasia or tumor.In some embodiments, alteration of methylation implies an increase or decrease of 5hmC. In some embodiments, alteration of methylation implies a lower incidence of 5hm C. The shores of CpG Island may be located in various regions in relation to the CpG Islands (see, eg, Irizarry et al. (2009) Nat. Genetics 41; 178-186). In some embodiments, the CpG island shores are located at a distance of less than 100 bp; 100-250 bp; 250-500 bp; 500-1000 bp; 1000-1500 bp; 1500-2000 bp; 2000-3000 bp ; 3000 bp or more of a CpG Island.
[0096] As used herein, the term "metastasis" serves to refer to a process in which cancer cells originating in an organ or part of the body are relocated to another part of the body and continue to replicate. Metastatic cells subsequently form tumors that can continue to metastasize. Metastasis thus refers to the spread of cancer from the part of the body in which it originally occurs to other parts of the body.
[0097]
[0098] As used herein, "it is suspected that an individual is susceptible to metastasize cancer" serves to refer to an individual who is at risk above the average of developing a metastatic cancer. Among the examples of individuals in particular risk of developing cancer of a particular type (eg, colorectal cancer, gallbladder cancer, breast cancer, prostate cancer) include those whose family medical history indicates an above-average incidence of such cancer among family members and / or those who they have already developed cancer and have been treated effectively, which therefore face the risk of relapse or recurrence. Other factors that may contribute to an above-average risk of developing metastatic cancer that lead to classify an individual as suspected of being susceptible to metastatic cancer may be based on the specific genetic, medical and / or behavioral characteristics and background of the individual.
[0099] The term "neoplasm" as used herein refers to any abnormal and new tissue growth. Therefore, a neoplasm can be a premalignant neoplasm or a malignant neoplasm. The term "specific marker of neoplasm" refers to any biological material that can be used to indicate the presence of a neoplasm. Examples of biological materials include, without limitation, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (eg, cell membranes and mitochondria) and whole cells.
[0100]
[0101] As used herein, the term "amplicon" refers to a nucleic acid generated by the use of primer pairs. The amplicon is usually single-stranded DNA (eg, the result of asymmetric amplification), although it may be RNA or cDNA.
[0102]
[0103] The term "amplify" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide or a portion of the polynucleotide, usually starting from a small amount of the polynucleotide (eg, a single molecule of polynucleotide), in which amplification products or amplicons are generally detectable. Polynucleotide amplification encompasses various chemical and enzymatic processes. Generation of multiple copies of DNA from one or a few copies of a target or matrix DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (CSF; see, e.g. ., U.S. Patent No. 5,494,810) are forms of amplification. Other types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611), Assembly PCR (see, e.g., U.S. Patent No. 5,965. 408), helicase-dependent amplification (see, e.g., U.S. Patent No. 7,662,594), hot start amplification (see, e.g., U.S. Patent Nos.
[0104] 5,773,258 and 5,338,671), Intersection-specific PCR, reverse PCR (see, eg, Triglia, et al. (1988) Nucleic Acids Res., 16: 8186), ligation-mediated PCR (see, p .ej., Guilfoyle, R. et al., Nucleic Acids Research, 25: 1854-1858 (1997); U.S. Patent No. 5,508,169), methylation-specific PCR (see, eg, Herman, et al. ., (1996) PNAS 93 (13) 9821-9826), PCR minicerator, multiplex ligation-dependent probe amplification (see, eg, Schouten, et al., (2002) Nucleic Acids Research 30 (12): e57), Multiplex PCR (see, eg, Chamberlain, et al., (1988) Nucleic Acids Research 16 (23) 11141-11156; Ballabio, et al., (1990) Human Genetics 84 (6) 571- 573; Hayden, et al., (2008) BMC Genetics 9: 80), nested Pc R, overlapping extension PCR (see, eg, Higuchi, et al., (1988) Nucleic Acids Research 16 (15) 7351-7367), real-time PCR (see, e.g., Higuchi, et al. (1992) Biotechnology 10: 413-417; Higuchi, et al. (1993) Biotechnology 11: 1026-1030), PCR d and reverse transcription (see, e.g., Bustin, S.A. (2000) J. Molecular Endocrinology 25: 169-193), Solid Phase PCR, Asymmetric Interlacing Thermal PCR and Touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19 ( 14) 4008; Roux, K. (1994) Biotechniques 16 (5) 812-814; Hecker, et al., (1996) Biotechniques 20 (3) 478-485). Polynucleotide amplification can also be carried out using digital PCR (see, eg, Kalinina, et al., Nucleic Acids Research. 25; 1999 2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci United States 96; 9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525).
[0105]
[0106] As used herein, the terms "complementary" or "complementarity" are used by referring polynucleotides (ie, a nucleotide sequence) related by the rules of base pairing. For example, the sequence "5'-A-G-T-3 '," is complementary to the sequence "3'-T-C-A-5'". Complementarity can be "partial," in which only some of the nucleic acid bases are paired according to the base pairing rules. Or, there may be "complete" or total "complementarity between nucleic acids. The degree of complementarity between nucleic acid chains has a significant effect on the efficacy and intensity of hybridization between nucleic acid chains. This is particularly important in amplification reactions, as well as detection procedures that depend on the binding between nucleic acids.
[0107]
[0108] As used herein, the term "primer" refers to an oligonucleotide, whether of natural origin, as in a purified restriction digest, or produced synthetically, that is capable of acting as a starting point for the synthesis when the conditions under which the synthesis of a primer extension product is induced which is complementary to a nucleic acid chain (e.g., in the presence of nucleotides and an induction agent such as a biocatalyst, can be induced) (e.g., a DNA polymerase or the like) and at a suitable temperature and pH). The primer is normally single stranded for maximum efficiency in amplification, although, alternatively, it can be double stranded. If it is double stranded, the primer is first treated to separate its chains before using it to prepare extension products. In some embodiments, the primer is an oligodeoxyribonucleotide. The primer is long enough to stimulate the synthesis of extension products in the presence of the induction agent. The exact lengths of the primers will depend on many factors, including the temperature, the source of the primer and the use of the procedure. In certain embodiments, the primer is a capture primer.
[0109]
[0110] As used herein, the term "nucleic acid molecule" refers to any molecule that contains nucleic acid, including, but not limited to, DNA or RNA. The term encompasses sequences that they include any of the DNA and RNA base analogs, including, but not limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine, acyridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5- bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methyl-guinene, 2,2-dimethyl-guanine, 2,2-dimethyl-guanine methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, b-glucosyl-5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxy-amino-methyl-2-thiouracil, beta-D-mannosylkeosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- Thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine and 2,6-diaminopurine.
[0111]
[0112] As used herein, the term "nucleobase" is synonymous with other thermals used in the art, including "nucleotide," "deoxynucleotide," "nucleotide moiety," "deoxynucleotide moiety," "nucleotide triphosphate (NTP) , "or deoxynucleotide triphosphate (dNTP).
[0113]
[0114] An "oligonucleotide" refers to a nucleic acid that includes at least two nucleic acid monomer units (eg, nucleotides), usually more than three monomer units and more normally more than ten monomer units. The exact size of an oligonucleotide generally depends on several factors, including the function or ultimate use of the oligonucleotide. For further illustration, oligonucleotides are normally less than 200 residues in length (eg, between 15 and 100), however, as used herein, it is intended that the term also encompasses more polynucleotide chains. long Reference is usually made to oligonucleotides according to their length. For example, an oligonucleotide of 24 is referred to as that of "24-mer". Normally, the nucleoside monomers are linked by phosphodiester bonds or analogs thereof, including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoranilidate, phosphoramidate and the like, including associated counter-ions, e.g., H +, NH4 +, Na + and the like, if said counterions are present. Also, oligonucleotides are normally single stranded. Optionally, oligonucleotides are prepared by any suitable procedure, including, but not limited to, isolation of an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or chemical synthesis. direct through a procedure such as the phosphotriester process of Narang et al. (1979) Meth Enzymol. 68: 90-99; the phosphodiester procedure of Brown et al. (1979) Meth Enzymol. 68: 109-151; the diethylphosphoramidite procedure of Beaucage et al. (1981) Tetrahedron Lett. 22: 1859-1862; the triester process of Matteucci et al. (1981) J Am Chem Soc. 103: 3185-3191; automatic synthesis procedures; or the solid support procedure of US Patent No. 4,458,066, entitled "POLINUCLEOTID PREPARATION PROCEDURE," published July 3, 1984 to Caruthers et al., or other procedures known to persons skilled in the art.
[0115]
[0116] A "sequence" of a biopolymer refers to the order and identity of polymer units (eg, nucleotides, etc.) in the biopolymer. The sequence (eg, base sequence) of a nucleic acid is normally read in the 5 'to 3' direction.
[0117] As used herein, the term "subject" refers to any animal (eg, a mammal), including, but not limited to, humans, non-human primates, rodents and the like, which will be the recipient of a particular treatment. Normally, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject.
[0118]
[0119] The term "gene" refers to a nucleic acid sequence (eg, DNA) that comprises coding sequences necessary for the production of a polypeptide, RNA (eg including, but not limited to, mRNA, TRNA and rRNA) or precursor. The polypeptide, RNA, or precursor may be encoded by a full length coding sequence or by a portion of the coding sequence as long as the desired activity or functional properties are retained (e.g., enzymatic activity, binding of ligand, signal transduction, etc.) of the full length or fragment. The term also encompasses the coding region of a structural gene and the included sequences located adjacent to the coding region at both ends 5 'and 3' for a distance of approximately 1 kb at each end, so that the gene corresponds to the full length mRNA length. Sequences that are located in the 5 'direction of the coding region and that are present in the mRNA are called 5' untranslated sequences. Sequences that are located in the 3 'direction or downstream of the coding region and that are present in the mRNA are called 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genetic form, or a clone of a gene, contains the interrupted coding region with non-coding sequences that are called "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into nuclear RNA (RNAn); introns may contain regulatory elements, such as enhancers. Introns are separated or "unpacked" from the nuclear or primary transcript, introns are therefore absent in the transcript processed by messenger RNA (mRNA). The mRNA works during translation to specify the sequence or order of amino acids in a nascent polypeptide.
[0120]
[0121] Detailed description of the invention
[0122]
[0123] The present invention relates to methods and kits for detecting 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5meC). In some embodiments, the present invention relates to the detection of 5hmC in genomic DNA, eg, mammalian genomic DNA. The procedures currently available to identify 5hmC have a resolution limit of approximately 50-200 base pairs. Many of current procedures are limited by the bisulfite conversion stage that cannot distinguish between 5-methylcytosine (5meC) and 5hmC. The present invention addresses both problems. First, the present invention allows discriminating between 5meC and 5hmC DNA modifications. Secondly, the present invention allows the detection of both 5meC and 5hmC in single base resolution.
[0124]
[0125] The procedure described herein identifies 5-hydroxymethylcytosine (5hmC) in DNA with single base resolution. Additionally, said procedure can identify 5meC in a specific base resolution simultaneously with 5hmC. The procedure used takes advantage of the fact that DNMT1 methyl transferase cannot methylate transversely from a 5hmC (or modified 5hmC; as is the case with p-glucosyl-5-hydroxymethylcytosine) and preferably transverse methyl from 5-methylcytosine (5meC). After sequential rounds of a cycle of PCR amplification and treatment of DNA with DNMT1, the population of DNA containing 5hmC is diluted by a factor of two while the population containing 5meC remains stable. This dilution accompanied by the conversion with bisulfite allows the specific base identification of DNA residues containing 5hmC (Figure 1).
[0126]
[0127] DNA bisulfite conversion results in the conversion of unmodified cytosine (C) into uracil (U) that will be read as thymine (T) after DNA sequencing amplified by PCR. Both 5meC and 5hmC are protected against conversion and will not be converted to U. Therefore, both will be read as C after sequencing (see Figure 2). Conversion with bisulfite is a perfectly established technology considered for a long time as the reference criterion for the detection of 5meC and only recently (2010) has it been reported in the scientific literature that one cannot distinguish between 5meC and 5hmC30. However, the procedure described in this document takes advantage of this fact to create a set of reference data (with the reference "A" in Figure 1).
[0128]
[0129] In the present invention, 5hmC is diluted in the total DNA pool while maintaining 5meC. This dilution is achieved through sequential rounds of a PCR amplification cycle and treatment of the DNA with the maintenance methyl DNA transferase, DNMT1, which maintains enzymatic and specifically 5meC only by adding a methyl group to the unmethylated chain of the products of Hemimethylated PCR (in Figure 1 this sample is referred to as "B"). After one or more rounds of this test, bisulfite conversion is performed, followed by sequencing of the treated DNA sample, in which 5meC is now the predominant modification. It is contemplated that all or most of the bases read as C of this sample have been protected against conversion by 5meC and not by 5mC. By comparing with the reference sample "A" it is possible to detect all base positions containing 5hmC. Dilution can be achieved on a basis as broad as the genome or with respect to a particular gene locus or a portion of a gene. In preferred embodiments, the dilution region is defined by the primers used for replication and / or amplification of the region of target interest.
[0130]
[0131] Accordingly, in some embodiments, the present invention provides methods for detecting or determining the status of 5meC and 5hmC in a predetermined region of a genomic DNA sample. In some preferred embodiments, the predetermined region (or region of target interest) corresponds to a locus of the gene of interest or a portion of a gene. In some embodiments, the predetermined region is defined by the nucleic acid primers used for replication or amplification of the predetermined region.
[0132]
[0133] The nucleic acid sample is divided into at least two portions for further analysis. In some embodiments, the first portion is replicated under conditions such that the 5-methylated cytosine residues are maintained and the 5-hydroxymethylated cytosine residues are diluted. The present invention is not limited to any particular dilution level. For example, the 5-hydroxymethylated cytosine residues can be diluted by a factor of 1.5, 2, 5, 10, 20, 40, 100, 200, 400, 800, 1600 or more.
[0134]
[0135] In some embodiments, dilution of 5-hydroxymethylated cytosine residues is carried out by replication of the nucleic acid (preferably replication of the predetermined region) with a polymerase to provide replicated nucleic acid and then treatment of the replicated nucleic acid with an enzyme that add a methyl group to the unmethylated chain of hemimethylated nucleic acid, but do not add a hydroxymethyl group to the non-hydroxymethyl chain of hemihydroxymethylated nucleic acid. The present invention is not limited to the use of a particular enzyme. In some embodiments, the enzyme is an enzyme that maintains the DNA methylation state of a nucleic acid, for example a methyl transferase DNA (DNMT). Examples of DNA methyl transferases include, but are not limited to, mouse DNMT1 (SEQ ID NO: 1; Figure 7), human Dn Mt 1 (SEQ ID NO: 2, Figure 8) or M.SssI DNMT (Spiroplasma sp.) (SEQ ID NO: 3, Figure 9) or a homologue or variant thereof. In some embodiments, homologs or variants have the activity of adding a methyl group to the unmethylated chain of a hemimethylated nucleic acid. In some embodiments, homologs or variants have at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity with SEQ ID NO: 1, 2 or 3 and / or have the activity of adding a methyl group to the unmethylated chain of a hemimethylated nucleic acid.
[0136]
[0137] In some embodiments, the replication step is performed through one or more rounds of chain reaction of polymerase In preferred embodiments, a predetermined region is replicated by extension from nucleic acid primers defining the 5 'and 3' ends of the predetermined region. The replicated nucleic acid is then treated with a DNA methylation enzyme, as described, to maintain 5-methylcytosine methylation of the predetermined region and then the procedure is repeated until the desired level of dilution of cytosine residues is achieved. 5-hydroxymethylated compared to 5-methylated moieties. In some embodiments, the dilution level per cycle is preferably approximately double, but may be as low as 1.1. In some embodiments, the maintenance level of 5-methyl cytosine residues is approximately 100%, but it can be up to 10% and continue to provide effective determination and discrimination between the 5meC and 5hmC residues in the predetermined region. In some embodiments, the number of replication cycles and treatment with DNA methylation enzyme may be 1, 2, 3, 5, 7, 10 or 20 cycles or more, or between about 1 and 20 cycles.
[0138] In some embodiments, labeled primers are used in the replication stage in order to be able to isolate labeled extension products from the replication stage using a marker binding reagent and use them in the later stages, such as for treatment with methyl DNA. transferase In preferred embodiments, only the newly synthesized chains (ie, chains marked by the labeled primer) are used and analyzed in the subsequent steps. Figure 11 provides a schematic representation of the use of labeled primers in the procedure. In said Figure, "A" presents the conventional bisulfite conversion and sequencing test and "B" presents the methyl transferase-dependent assay. As shown in the left panel for the "B" test, the use of primer extension from the biotinylated primer and subsequent isolation with streptavidin beads ensures that all the lower chains in the analysis are newly synthesized. Therefore, performing the treatment with DNMT1 (or other methyl transferase) and then analyzing the lower chains isolated with biotin-streptavidin will achieve a direct and precise quantification of the 5meC level of the complementary chain. In the panel on the right, in the upper part, the sequencing results representative of 10 clones for the conventional bisulfite test, "A", in which both 5meC and 5hmC as cytosine after the treatment are read, and the Representative sequencing results of 10 clones for the "B" methyl transfer assay in which only 5meC will be read as cytosine after treatment. The combination of the conventional bisulfite test data "A", in which both 5meC and 5hmC as cytosine are read after treatment and the methyl transfer test "B" in which only 5meC is read as cytosine after treatment allows to determine the position and quantity of 5hmC (from a simple calculation: AB = 5hmC). This quantification is indicated in the lower part of the right panel. For experimental replicates with this same quantitative result, n = 15 was obtained.
[0139]
[0140] The present invention is not limited to the use of any labeled primer or particular label binding reagent for the isolation of the labeled primer. In some preferred embodiments, the primer is biotinylated and the marker binding reagent is a streptavidin reagent, such as a streptavidin bead. Replicated nucleic acid chains comprising the biotinylated primer (ie, the primer extension product resulting from the biotinylated primer extension) are isolated by contacting the chains with streptavidin beads. It is possible to use any combination of labeled primer and label binding reagent. Other suitable examples include haptenylated primers and beads or other reagents comprising an antibody or other antigen binding protein that binds to the hapten. Suitable haptens include, but are not limited to, pyrazoles, in particular nitropyrazoles; nitrophenyl compounds; benzofurazanos; triterpenes; ureas and thioureas, in particular phenyl ureas and even more particularly phenyl thioureas; rotenone and rotenone derivatives, which are also referred to herein as rotenoids; oxazole and thiazoles, particularly oxazole and thiazole sulfonamides; coumarin and coumarin derivatives; cycloolignans, such as podophyllotoxin and podophyllotoxin derivatives; and combinations thereof. Specific examples of haptens include, but are not limited to, 2,4-dintrophenyl (DNP), biotin, fluorescein derivatives (FITC, TAMRA, Texas Red, etc.), digoxigenin (DIG), 5-nitro- 3-pyrozolcarbamide (nitropyrazol, NP), 4,5, -dimethoxy-2-nitrocinamide (nitrocinamide, NCA), 2- (3,4-dimethoxyphenyl) -quinoline-4-carbamide (phenylquinolone, DPQ), 2,1, 3-benzoxadiazol-5-carbamide (benzofurazano, BF), 3-hydroxy-2-quinoxalincarbamide (hydroxyquinoline, HQ), 4- (dimethylamino) azobenzene-4'-sulfonamide (DABSIL), rotenone isoxazoline (Rot), (E) -2- (2- (2-oxo-2,3-dihydro-1 H -benzo [b] [1,4] diazepin-4-yl) phenoxy) acetamide (benzodiazepine, BD), 7- (diethylamino) acid - 2-oxo-2H-chromene-3-carboxylic acid (coumarin 343, CDO), 2-acetamido-4-methyl-5-thiazolsulfonamide (thiazolsulfonamide, TS) and p-methoxyphenylpyrazophopodofillamide (Podo).
[0141]
[0142] In some embodiments, the 5hmC groups of the sample are modified with the blocking group to increase the efficiency ratio of methyl transferase between 5meC and 5hmC. As used herein, a "blocking group" is any chemical group that can be added at 5hmC (or cytosine in the 5-carbon position) that makes the total group too large, or unfavorably charged. , for the pocket of DNA methyl transferase and thus block the activity of DNA methyl transferase in the remainder 5hmC. It is contemplated that the use of blocking groups increases the specificity and / or efficiency ratio of DNMT1 methyl transferase to catalyze the transfer of a methyl group transversely from 5meC and 5hmC in cDNA. The present invention is not limited to the use of any particular blocking group. Suitable blocking groups include but are not limited to Glucose (beta-glucose and alpha-glucose); Gentiobiosa (6-OpD-glucopyranosyl-D-glucose) (and any other stereoisomer, alpha binding is also possible: 6-O-alpha-D-glucopyranosyl-D-glucose); keto-glucose; azidaglucose (eg N3Glucose); a chemical group linked to glucose or azide-glucose, eg, by chemical click, for example biotin (biotin-N3Glucose-5hmC); JPB1 (J binding protein 1) linked to glu-5hmC (full length and truncated versions); TET proteins (eg TET1, TET2 and TET3) (full length and truncated versions) binding at 5hmC; other 5hmC or Glu-5hmC binding proteins and / or protein binding domains; (native and cross-linked versions of proteins); any glucose or modified glucose oxidation product, eg, oxidized glucose with periodate; any chemical group that reacts with oxidized glucose to bind or modify glucose; and any protein or protein complex that can specifically identify 5meC, 5hmC and modified variants of these bases (eg, JPB1 and MPB class proteins (eg, MPB1 and MeCP2)).
[0143]
[0144] Without blocking it is possible to achieve 100% versus 0% versus 0% methyl transfer transversely from 5meC, C and 5hmC respectively, although the procedure is also applicable to less than 100% methyl transfer transversely from 5meC and more than 0% transverse transfer from C and 5hmC. In these cases, greater quantification accuracy can be obtained when a known control is added to the sample in order to determine the efficiency in the sample. With blocking it is possible to achieve 100% versus 0% versus 0% methyl transfer transversely from 5meC, C and 5hmC respectively, although the procedure can be applied at less than 100% methyl transfer transversely from 5meC and more than 0% transfer transversely from C and 5hmC. Blocking can be useful for the "conventional" test because it will allow to obtain more solid 100% versus 0% versus 0% with the DNMT1 test with a higher success rate compared to the lack of blocking.
[0145]
[0146] With blocking, 100% versus 100% versus 0% methyl transfer can be achieved transversely from 5meC, C, 5hmC respectively for M.SssI (or DNMT1, preferably a large molar excess of DNMT1). Methylation transversely from 5meC and C is an alternative way of transferring the modification status information from the parental chain to the replicated / extended primer chain to help identify the positions and quantities of 5meC, 5hmC and C. This may allow Direct reading of 5hmC as unmodified cytosines that are not protected from bisulfite conversion or, in comparison to conventional bisulfite conversion and sequencing, can reveal quantitative information for 5meC, C and 5hmC in the nucleic acid sequence. This will allow to reveal through a simple calculation the position and quantity of 5meC, C and 5hmC.
[0147]
[0148] It is likely that the identification of 5meC, 5hmC and C can be achieved if the blockade is carried out in the 5hmC and C residues. The blocking agents in the cytosine residues could be for example proteins containing the CXXC motif or any protein or fragment of the same that can be attached to CpG without modifying.
[0149]
[0150] In some embodiments, the diluted 5hmC nucleic acid sample and the undiluted portion are treated to convert unmodified cytosine residues to thymidine residues. In preferred embodiments, the portions are treated with bisulfite to convert unmodified cytosine residues into uracil residues. The bisulfite treated nucleic acid is then replicated with a polymerase to convert said uracil residues to thymidine residues. In some embodiments, the replication step is performed through one or more rounds of polymerase chain reaction (see, e.g., Figures 1 and 5) or primer extension reaction (see, e.g. , Fig. 5). In preferred embodiments, a predetermined region is replicated by extension from nucleic acid primers defining the 5 'and 3' ends of the predetermined region. In some embodiments, the number of replication cycles may be greater than 2, 3, 5, 7, 10 or 20 cycles or between approximately 2 and 20 cycles.
[0151]
[0152] The procedure described in the preceding paragraphs provides two different portions of nucleic acid. In the first portion, the 5-hydroxymethylated moieties have been diluted compared to the 5-methylated cytosine moieties, which have been maintained. In the second portion, the 5-hydroxymethylated residues have not been diluted. When the bisulfite portions are treated, all unmodified cytosine residues are converted into uracil residues and then thymidine residues after 1 or more rounds of replication or primer extension. In preferred embodiments, both portions are sequenced, preferably using primers that allow sequencing of the predetermined region. In preferred embodiments, the comparison of the sequences of the first and second portions allows the identification of 5meC and 5hmC residues in the predetermined region. 5hmC residues are identified as residues that are read by sequencing as a thymidine residue in the first portion (i.e., the portion in which the 5hmC residues have been diluted) and as a cytosine residue in the corresponding position in the second portion of nucleic acid and residues of 5meC residues are identified as residues that are read as cytosine in both the first and second portions of the nucleic acid.
[0153]
[0154] Sequencing of nucleic acid samples can be performed through any of the procedures known in the art. Suitable sequencing procedures include, but are not limited to, chain termination sequencing procedures (eg, Sanger sequencing procedures) and second generation DNA sequencing procedures in which the systems are used. provided by Illumina (San Diego CA), Pacific Biosciences (Menlo Park, CA) and others. In embodiments in which second generation sequencing procedures are used, the step of replication with the polymerase before sequencing (which converts the uracil moiety to a thymidine moiety) is optional and the uracil moiety can be read directly.
[0155]
[0156] In some embodiments, the procedures described are used to predict a predisposition of a subject to contract a disease, diagnose a disease in a subject, predict the likelihood of recurrence of a disease in a subject, provide a diagnosis for a subject who has a disease. or select a subject suffering from a disease for treatment with a particular therapy. Such methods preferably comprise providing a genomic DNA sample of a subject; and detect the status of mutilation of predetermined regions of the genomic DNA sample through the procedures described. In some embodiments, an alteration of the level of methylation of 5-hydroxymethylcytosine and / or 5-methylcytosine (ie, a greater or lesser level) of the predetermined regions of the genomic DNA with respect to a state of reference methylation provides an indication selected from the group consisting of an indication of a predisposition of the subject to a disease, an indication that the subject has a disease, an indication of the likelihood of recurrence of a disease in the subject, an indication of the subject's survival and an indication that the subject is a candidate for treatment with a particular therapy.
[0157]
[0158] Consequently, in some embodiments, the methods of the present invention involve the determination (eg, evaluation, assessment, quantification) of the level of modification 5meC and / or 5hmC of an indicator of a condition of interest, such as neoplasm. , in a sample. Persons skilled in the art understand that a level of modification 5meC and / or 5hmC greater, lesser, informative or otherwise clearly distinguishable is articulated with respect to a reference (eg, a reference level, a level of control, a threshold level or similar). For example, the expression "high level of 5hmC or 5meC", as used herein with respect to the state of 5hmC or 5meC of a gene locus is any level of 5hmC and / or 5meC that is above the median level of 5hmC or 5meC in a sample of a random population of mammals (e.g., a random population of 10, 20, 30, 40, 50, 100 or 500 mammals) that do not have a neoplasm (e.g. ., a cancer) or other condition of interest. High levels of modification 5meC and / or 5hmC can be any level as long as the level is higher than the corresponding reference level. For example, a high level of 5meC and / or 5hmC of a locus of interest can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, times more than the reference level of 5meC and / or 5hmC observed in a normal sample. It should be noted that the reference level can be any amount. The expression "high 5meC and / or 5hmC score", as used herein with respect to the 5meC and / or 5hmC events detected in a particular nucleic acid marker matrix panel is any 5meC score. and / or 5hmC that is above the median of the 5meC and / or 5hmC score in a sample of a random population of mammals (eg, a random population of 10, 20, 30, 40, 50, 100 or 500 mammals) that do not have a neoplasm (eg, a cancer) A high score of 5hmC in a particular matrix panel of nucleic acid markers can be any score as long as the score is higher than that of the corresponding reference score For example, a high score of 5meC and / or 5hmC in a locus of interest can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times above the reference score of 5meC and / or 5hmC in a normal sample. It should be noted that the pu Reference rate can be any quantity used for comparative purposes.
[0159]
[0160] Similar considerations apply for tests of decreasing levels of modifications of 5meC and / or 5hmC in a sample, target locus, target genomic region and the like. For example, the expression "decrease in the level of 5meC and / or 5hmC" as used herein with respect to the state of 5meC and / or 5hmC of a gene locus is any level of 5meC and / or 5hmC that is below the median level of 5meC and / or 5hmC in a sample of a random population of mammals (eg, a random population of 10, 20, 30, 40, 50, 100 or 500 mammals) that does not have a neoplasm (eg, a cancer). The decrease of the 5meC and / or 5hmC modification levels can be any level as long as the level is lower than the corresponding reference level. For example, a decrease in the level of 5meC and / or 5hmC of a locus of interest may be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times below the level 5meC and / or 5hmC reference observed in a normal sample. It should be noted that a reference level can be any amount. The expression "lower 5hmC score", as used herein with respect to the 5meC and / or 5hmC events detected in a particular nucleic acid marker matrix panel is any 5meC and / or 5hmC score. that is below the median of the 5meC and / or 5hmC score in a sample of a random population of mammals (eg, a random population of 10, 20, 30, 40, 50, 100 or 500 mammals) who do not have a neoplasm (eg, a cancer). A lower score of 5meC and / or 5hmC in a particular nucleic acid marker matrix panel can be any score as long as said score is greater than the corresponding reference score. For example, a score less than 5meC and / or 5hmC in a locus of interest may be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times below the 5meC and / or 5hmC reference score observed in a normal sample. It should be noted that the reference score can be any quantity that is used for comparative purposes.
[0161]
[0162] The procedures are not limited to a particular type of mammal. In some embodiments, the mammal is a human being. In some embodiments, the neoplasm is premalignant. In some embodiments, the neoplasm is malignant. In some embodiments, the neoplasm is cancer regardless of the stage (eg, stage I, II, III or IV).
[0163] The present disclosure also provides procedures and materials to help medical and research professionals determine whether or not a mammal suffers from a neoplasm (eg, cancer). Medical professionals can be for example doctors, nurses, medical technicians and laboratory pharmacists. Research professionals can be, for example, principal researchers, research technicians, postdoctoral interns and postgraduate students. You can help a professional (1) by determining the ratio of 5hmC and / or other markers in a sample and / 2) by communicating information about the relationship to that professional, for example.
[0164]
[0165] Once the level (eg, score or frequency) of a 5meC and / or 5hmC modification in particular in a sample has been notified, the medical professional may take one or more actions that affect the medical care of the patient. For example, a medical professional can record the results in the patient's medical history. In some cases, a medical professional may record a diagnosis of neoplasia or otherwise transform the patient's medical history to reflect the patient's pathology. In some cases, a medical professional can review and evaluate the patient's complete medical history and evaluate various treatment strategies for the clinical intervention of a patient's condition. In some cases, a medical professional may record the prediction of a tumor with other reported indicators. In some cases, a medical professional can review and evaluate the patient's medical history in its entirety and evaluate various treatment strategies for the clinical intervention of a patient's condition.
[0166]
[0167] A medical professional can initiate or modify the treatment of a neoplasm once the information is received regarding the level (score, frequency) associated with the level of 5meC and / or 5hmC in a patient's urine sample. In some cases, a medical professional may compare previous reports and the level (score, frequency) of modification of 5meC and / or 5hmC that has been reported recently and recommend a change in therapy. In some cases, the medical professional may recruit a patient for a clinical trial for a new therapeutic intervention of neoplasm. In some cases, a medical professional may choose to wait to start therapy until the patient's symptoms require clinical intervention.
[0168]
[0169] A medical professional can communicate the test results to the patient or a patient's family member. In some cases, a medical professional may provide information related to the neoplasm to the patient and / or a patient's family member, including treatment options, a prognosis or referral to specialists, eg, oncologists and / or radiologists. In some cases, a medical professional may provide a copy of the patient's medical history to communicate the test results to the specialist. A research professional may request information on the results of the subject's trial to advance the neoplasm investigation. For example, a researcher may collect data on the results of the trial with information about the efficacy of a drug for the treatment of neoplasia to identify an effective treatment. In some cases, a research professional may obtain the test results to assess the recruitment of a subject or continued participation in a research study or clinical trial. In some cases, a research professional can classify the severity of a subject's condition, based on the test results. In some cases, a research professional may communicate the results of the subject's test to a medical professional. In some cases, a research professional may refer a subject to a medical professional for the clinical evaluation of a neoplasm and its treatment. An appropriate procedure can be used to communicate the information to another person (eg, a professional). For example, the information can be provided directly or indirectly to a professional. For example, a laboratory technician can enter the test results into a computer register. In some cases, the information is communicated by introducing a physical alteration in the clinical or research records. For example, a medical professional can make a permanent medical record or mark a medical history to communicate a diagnosis to other medical professionals who review that history. On the other hand, any type of communication can be used to transmit the information. For example, physical mail, e-mail, telephone and face-to-face interactions. It is also possible to transmit the information to a professional by providing the professional with the information electronically. For example, it is possible to transmit the information to a professional by entering the information into a computer database so that the professional can access that information. On the other hand, it is possible to communicate the information to a hospital, clinic or research center as an agent for the professional.
[0170]
[0171] It should be noted that it is possible to analyze the sample for a specific marker of neoplasm or for several specific markers of neoplasm. In preferred embodiments, a single sample is analyzed for several specific neoplasm markers, for example, using multi-marker assays. On the other hand, it is possible to extract several samples from a single mammal and analyze them as described herein. In some embodiments, a sample is divided into a first and a second portion, in which the first portion is subjected to cytological analysis and the second portion to purification or other treatments (eg, specific capture stage (s) of sequence (eg for the isolation of specific loci for the analysis of 5hmC levels.) In some embodiments, the sample is subjected to one or more steps of the procedure before dividing it into portions. In some embodiments, it is treated, manipulate or preserve the sample so that DNA integrity is promoted and / or DNA degradation is inhibited (e.g. through the use of storage buffers with stabilizing agents (e.g., chelating agents, inhibitors) DNase) or manipulation or treatment techniques that promote DNA integrity (e.g., immediate treatment or low-temperature storage (e.g., -80 degrees C)).
[0172]
[0173] In some embodiments, all essential materials and basic reagents for detecting neoplasia are assembled in a kit by detecting the level (presence, absence, score, frequency) of markers in a sample obtained from a mammal, as defined in claim 14. Said kits generally comprise, for example, useful reagents, sufficient or necessary to detect and / or characterize one or more markers (eg, epigenetic markers; 5hmC modifications) specific for a neoplasm. In some embodiments, the kits contain enzymes suitable for amplifying nucleic acids including various polymerases, deoxynucleotides and buffers to provide the reaction mixture necessary for amplification. In some embodiments, the kits of the present invention include a means for containing reagents confined in the space for commercial sale, such as, for example, blow molded or injection molded plastic containers in which the desired reagent is retained. . Other suitable packages are also provided to carry out the specific stages of the disclosed processes.
[0174] In some embodiments, the procedures disclosed herein are useful for tracking the treatment of neoplasia (eg, cancer). For example, in some embodiments, the procedures can be carried out immediately before treatment, during and / or after treatment to track the success of the treatment. In some embodiments, the procedures are performed at intervals in patients without the disease to ensure the success of the treatment.
[0175]
[0176] The present disclosure also provides a series of embodiments with computer support. Specifically, in some embodiments, the invention provides a computer program for analyzing and comparing the results of a neoplasm specific marker detection pattern in a sample obtained from a subject, for example, with a library of said marker patterns recognized as indicative. of the presence or absence of a neoplasm or a particular stage or neoplasm. The present disclosure provides a computer program to analyze and compare a first and a second pattern of detection results of a specific marker of neoplasm from a sample taken at least at two different time points. In some parts of the disclosure, the first pattern may be indicative of a pre-cancerous condition and / or a low-risk state for cancer and / or progress from a pre-cancerous state to a cancerous state. In these parts, the comparison provides a follow-up of the progress of the condition from a first time point to the second time point.
[0177]
[0178] Elsewhere, the disclosure provides a computer program to analyze and compare a pattern of the detection results of the specific neoplasm marker from a sample with a library of specific neoplasm marker patterns recognized as indicative of the presence or absence of a cancer, in which the comparison provides for example a differential diagnosis between a benign neoplasm and an aggressively malignant neoplasm (eg, the marker pattern provides staging and / or graduation of the cancerous state).
[0179]
[0180] The procedures and systems described in this document can be implemented in various ways. In one part of the disclosure, the procedures involve the use of a communication infrastructure, such as the Internet. Several parts of the disclosure are explained below. It should also be understood that the present disclosure can be implemented in various forms of hardware, software, firmware, processors, servers (e.g., as used in computing at nine) or a combination thereof. The procedures and systems described in this document are implemented as a combination of hardware and software. The software can be implemented as an application program, tangibly specified in a computer storage device, or different parts of a software implemented in the user's computer environment (e.g., an applet) and in the reviewer's computer environment, being able to the reviewer is located in a remote location (eg, a service center of the provider).
[0181]
[0182] For example, it is possible to process the data in the computing environment of the user part while the user enters the data or after entering it. For example, the user's computing environment can be programmed to provide defined test codes to represent a platform, bearer / diagnostic test or both; data processing using defined indicators and / or generation of indicator configuration, the responses being transmitted as processed or partially processed in the reviewer's computing environment in the form of test code or indicator configurations for the subsequent execution of one or more algorithms to provide a result and / or generate a report of the reviewer's computing environment.
[0183]
[0184] The application program for executing the algorithms described herein can be loaded and executed through a machine comprising any suitable architecture. In general, the machine involves a computer platform that has a hardware, such as one or more central processing units (CPU), a random access memory (RAM) and input / output (I / O) interfaces. The computer platform also includes an operating system and a microinstruction code. The various procedures and functions described herein may be part of the microinstruction code or may be part of the application program (or a combination thereof) that are executed through the operating system. On the other hand, other peripheral devices can be connected to the computer platform, such as an additional data storage device or a printer.
[0185]
[0186] As a computer system, the system generally includes a processing unit. The processing unit functions to receive information, which generally includes test data (eg specific gene products tested) and test result data (eg, the pattern of specific marker detection results of neoplasm (eg epigenetic marker, 5hmC modifier) of a sample). The information received can be stored at least temporarily in a database and the data analyzed by comparing them with a library of marker patterns recognized as indicative of the presence or absence of a pre-cancerous state, or recognized as indicative of a stage and / or degree of cancer
[0187]
[0188] It is also possible to electronically send part or all of the input and output data; It is possible to send electronically or by telephone (eg, by fax, eg, using devices such as a fax) by virtue of certain output data. Examples of output receiving devices include a display element, a printer, a fax device and the like. Electronic forms of transmission and / or display on screen include email, interactive television, and the like. In some parts of the disclosure, they are kept on a server all or a portion of the input data and / or all or a portion of the output data (eg, usually, at least the library of neoplasm specific marker detection result patterns recognized as indicative of the presence or absence of a pre-cancerous state) for access, eg confidential access. The results can be sent or made accessible to professionals, as desired.
[0189]
[0190] A system for use in the procedures described herein generally includes at least one computer processor (e.g., in cases where the procedure is carried out entirely in a single location) or at at least two networked computer processors (e.g., in cases where a user enters the data of the detected marker for a sample obtained from a subject (e.g., a technician or the person performing the tests) and it is transmitted to a second computer processor at a remote location for analysis (eg, when the detection results of the pattern of the specific neoplasm marker are compared with a library of patterns recognized as indicative of the presence or absence of a pre-cancerous state), the first and second computer processors being connected by a network (eg, by intranet or internet) The system may include a user component (s) for input; and a component (s) of the reviewer to review the data and generate reports, including detection of a pre-cancerous state, staging and / or graduation of a neoplasm or monitoring the progress of a pre-cancerous state or a neoplasm. Other system components may include a server component (s); and a database (s) for storing data (eg, a database of report elements, eg a library of marker patterns recognized as indicative of the presence or absence of a pre-cancerous state and / or recognized as indicative of a grade and / or stage of a neoplasm or a relational database (BDR) that may include user data entry and exit data.Computer processors may be processors normally found in computers personal (eg, IBM, Dell, Macintosh), laptops, central computers, minicomputers or other computing devices.
[0191]
[0192] The input components can be complete, independent personal computers that offer the full range of power and features to run applications. The user component generally operates on any desired operating system and includes a communication element (e.g., a modem or other hardware for connection to the network), one or more input devices (e.g., a keyboard, a mouse, a numeric keypad or other device used to transfer information or commands), a storage item (e.g., a hard drive or a storage medium with computer support for writing and reading) and a display item (e.g., a monitor, a television, LCD, LED or other display device that converts information for the user). The user enters the input commands into the computer processor through an input device. Generally, the user interface is a graphical user interface (GUI) written for browser applications.
[0193]
[0194] The server component (s) can consist of a personal computer, a microcomputer and a central computer or they can be distributed across multiple servers (eg, as cloud computing applications) and offers data management , information shared between clients, administration and network security. The application and the databases used can be on the same or different servers. Other computer arrangements are also contemplated for the user and the server (s) including processing on a single machine, such as a central computer, a collection of machines, or other suitable configuration. In general, the user's machines and the server work together to carry out the processing of the present invention.
[0195]
[0196] When used, the database (s) is normally connected to the database server component and can be any device that holds data. For example, the database can be any optical or magnetic storage device for a computer (eg, CDROM, internal hard disk, a tape drive). The database can be located remotely from the server component (with network access, modem, etc.) or locally with respect to the server component.
[0197]
[0198] When used in the system and procedures, the database can be a relational database that is organized and accessed according to the relationships between the items in the data. The relational database is generally composed of several tables (entities). The rows of a table represent the records (collections of information about items separately) and the columns represent the fields (particular attributes of a record). In its simplest conception, the relational database is a collection of data entries that "keep a relationship" with each other through at least one common field.
[0199]
[0200] Additional workstations equipped with computers and printers can be used at the service point to enter the data and, in some parts of the disclosure, generate appropriate reports, if desired. The computer (s) may have a shortcut (eg, on the desk) to launch the application to facilitate the start of data entry, transmission, analysis, report reception, etc., if desired.
[0201]
[0202] The present disclosure is useful both for diagnosing diseases and disorders in a subject and for determining the prognosis of the subject. The procedures, reagents and systems of the present disclosure can be applied to a wide variety of diseases and disorders. Certain parts of the disclosure provide procedures for obtaining the risk profile of a subject from developing a neoplasm (eg, cancer). In some parts of the disclosure, such procedures involve obtaining a sample of a subject (eg, a human being at risk of developing cancer; a human being who is routinely tested), the Detection of the presence, absence or level (e.g., frequency or 5hmC modification score) of one or more specific markers for a neoplasm in the sample or associated with it (eg, specific for a neoplasm) in the sample and the generation of a risk profile of developing neoplasm (eg, cancer) based on the level detected (score, frequency) of the presence or absence of neoplasm indicators. For example, in some parts of the disclosure, the risk profile generated will change depending on the specific markers and that are detected as present or absent or at defined threshold levels. This disclosure is not particularly limited in the way of generating the risk profile. In some parts of the disclosure, a processor (eg, computer) is used to generate said risk profile. In some parts of the disclosure, the processor uses a specific algorithm (eg, software) to interpret the presence or absence of specific 5hmC modifications, as determined by the methods of the present invention. In some parts of the disclosure, the presence and absence of specific markers, as determined by the procedures of the present disclosure are entered into said algorithm and the risk profile is recorded based on a comparison of said entry with the established standards (eg, established pre-cancerous state norm, established norm for various levels of risk for developing cancer, established norm for subjects diagnosed with different stages of cancer). In some parts of the disclosure, the risk profile indicates a risk of the subject of developing cancer or a risk of the subject of developing cancer again. In some parts of the disclosure, the risk profile indicates for example a subject that has a very low, low, moderate, high or very high chance of developing or developing cancer again. In some parts of the disclosure, the healthcare professional (eg, an oncologist) will use that risk profile to determine the course of treatment or intervention (eg, biopsy, waiting, referral to an oncologist, referral to the surgeon, etc.).
[0203] Other diseases and disorders that can be diagnosed or predicted with the procedures, reagents and systems of the present disclosure include, but are not limited to, Prader-Willi syndrome, Angelman syndrome, Beckwith-Wiedemann syndrome, pseudohypoparathyroidism, Russell syndrome -Silver, ICF syndrome, Rett syndrome, a-thalassemia / mental retardation, X-linked (to TR-X), immune bone dysplasia, Schimke type, Rubinstein-Taybi syndrome, MTHFR deficiency, recurrent hydatidiform mole, syndrome of fragile X mental retardation, deletion of LCP and §P and 8p-thalassemia, FSH dystrophy, XIC disorder, Schimke's immune bone dysplasia (SIOD), Sotos syndrome, atrophy, X-linked Emery-Dreifuss muscular dystrophy (EDMD ), Autosomal EDMD, CMT2B1, mandibuloacral dysplasia, type 1B bone ring muscular dystrophy, familial partial lipodystrophy, dilated cardiomyopathy 1a, Hutchinson-Gilford progeria syndrome and Pelger-Huet anomaly.
[0204]
[0205] The following examples serve to demonstrate and better illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.
[0206]
[0207] Example 1
[0208]
[0209] The procedure described herein is based on the successive PCR dilution of the 5hmC modification together with the maintenance of the 5meC modification since DNMT1 cannot transverse from 5hmC and cytosine; however, DNMT1 can methylate DNA transversely from 5meC. Figures 3 and 4 demonstrate that DNMT1 cannot catalyze the transfer of a methyl group from S-adenosylmethylmethionine when the DNA substrate is a cytosine, 5hmC or p-glucosyl-5hmC. Therefore, it is possible to dilute the 5hmC modification by PCR followed by treatment with DNMT1 while maintaining the 5meC modification through multiple rounds of PCR and treatment with DNMT1.
[0210]
[0211] The process of the authors of the invention applies the conversion and sequencing with bisulfite of sample "A", untreated DNA, which will be used as a reference to detect the total of 5meC and 5hmC. The procedure involves a 5hmC dilution test, 5hmC dilution in the total cluster of DNA fragments while maintaining 5meC. The dilution is achieved through sequential rounds of a PCR amplification cycle (dilution) and DNA treatment with maintenance methyl DNA transferase, DNMT1, which maintains enzymatic and specifically 5meC by adding a methyl group only to the non-methylated chain of the Hemimethylated PCR products (in Figure 1, this sample is referred to as sample "B"). After a few rounds of this test, bisulfite conversion and sequencing of the treated DNA sample, sample B, in which 5meC is now the only modification present (or the only highly maintained modification) was applied. Therefore, all bases (or most of them) that are read as C from the sample must have been protected against conversion, by 5meC and not by 5hmC. By comparing "B" with this reference sample "A" it is possible to easily detect all base positions containing 5hmC.
[0212]
[0213] It should be noted that this procedure, while effectively diluting 5hmC, maintains the 5meC signal. Therefore, this procedure can serve two purposes (i) the identification of 5hmC in DNA and (ii) the identification of 5meC in DNA. The feasibility test of the described trial is demonstrated in Figure 4.
[0214]
[0215] Experimental design
[0216]
[0217] The procedure described herein is based on the successive PCR dilution of the 5hmC modification at the same time that the 5meC modification is maintained since DNMT1 cannot transverse methyl from 5hmC and cytosine; however, DNMT1 can methylate DNA transversely from 5meC. Figures 3. A and 4 demonstrate that DNMT1 cannot catalyze the transfer of a methyl group from S-adenosylmethylmethionine when the DNA substrate is a cytosine, 5hmC or b-glucosyl-5hmC. Therefore, it is possible to dilute the 5hmC modification by PCR followed by treatment with DNMT1 while maintaining the 5meC modification through multiple rounds of PCR and treatment with DNMT1. It should be noted that DNMT1 enzymes could be used, as well as other methyl transferases, to distinguish between 5meC and 5hmC even in cases in which these enzymes do methically transverse from 5hmC and cytosine as long as said enzymes have a preference for heme 5meC with respect to 5hmC, b-glucosyl-5hmC or cytosine at CpG sites (Figure 3. B and C). On the other hand, DNMT1 or the methyl transferase enzyme can allow the identification of both 5meC and 5hmC in the assay described herein even if the transfer of a methyl group from S-adenosyl methylmethionine is of a much lower rate than 100% . The requirement to distinguish 5meC from 5hmC is that there is a preference of 5meC with respect to 5hmC, b-glucosyl-5hmC or cytosine at CpG sites (Figure 3B and C).
[0218]
[0219] The process of the authors of the invention applies the conversion and sequencing with bisulfite of the sample "A", untreated DNA (Figure 1), which will be used as a reference since it will detect the total of both 5meC and 5hmC. The procedure involves a 5hmC dilution test, 5hmC dilution in the total cluster of DNA fragments while maintaining 5meC. The dilution is achieved through sequential rounds of a PCR amplification cycle (dilution) and DNA treatment with maintenance methyl DNA transferase, DNMT1, which maintains enzymatic and specifically 5meC by adding a methyl group only to the unmethylated chain of the Hemimethylated PCR products (in Figure 1, this sample is referred to as sample "B"). After a few rounds of this test, bisulfite conversion and sequencing of the treated DNA sample, sample B, was applied, in which 5meC is now the only modification present (or the only modification that is highly maintained). Therefore, all bases (or most of them) that are read as C from the sample must have been protected against conversion, by 5meC and not by 5hmC. By comparing "B" with the reference sample "A" it is possible to easily detect all base positions containing 5hmC.
[0220]
[0221] It should be noted that this procedure, while effectively diluting 5hmC, maintains the 5meC signal. Therefore, this procedure can serve two purposes (i) the identification of 5hmC in DNA and (ii) the identification of 5meC in DNA. The feasibility test of the described trial is demonstrated in Figure 4.
[0222]
[0223] On the other hand, it should be noted that bisulfite conversion kits have sensitivity limitations in the state of the art. For example, the MetilEasy Xceed kit (Human Genetic Signatures, cat. No. ME002) allows the 5meC analysis of only 8 cells, but does not allow a single cell analysis. The procedure described herein while diluting 5hmC and maintaining the 5meC signal, allows for greater detection sensitivity for both 5meC and 5hmC, with a clear potential for single cell analysis as a result of PCR amplification. of the DNA sample (with specific amplification of the gene or of the entire genome).
[0224]
[0225] Materials and Procedures
[0226]
[0227] Substrates
[0228]
[0229] DNA substrates created by hybridization of the appropriate complementary oligonucleotides (see Supplementary Table 1) by heating at 95 ° C and cooling at 1 ° C / min until the reaction reached 25 ° C. Oligonucleotides created by hybridization of higher 5hmC, upper 5meC or upper cytosine with lower cytosine were used in the methyl transferase specificity assay. The substrate used to simulate a round of PCR was created followed by treatment with DNMT1 by hybridization of 5hmC: C: 5meC higher with unmodified lower substrate. The substrate used for the complete assay was created by hybridization of 5hmC: C: 5meC higher with 5hmC: C: 5meC lower.
[0230] Methyl transferase specificity test
[0231]
[0232] Reactions (50 jl) containing 100 ng of DNA substrate (cytosine, hemi-5meC or hemi-5hmC), 50 mM Tris-HCl, 1 mM dithiothreitol, 1 mM EDTA pH 8.0, 5% (v / v) glycerin, S- [methyl-14C] -adenosyl-L-methionine at 37 ° C with 2 units of recombinant mouse DNMT1, recombinant human DNMT1 or SssI methyl transferase for 30 minutes. The reactions were terminated by the addition of 200 µl of TE buffer. The DNA of the reactions was precipitated with ethanol and washed three times with 70% ethanol cooled with ice. The DNA agglomerate was dried and suspended in 20 µl of TE buffer. The entire reaction was transferred to a 5 ml scintillation vial containing 2 ml of Ecosinct A. The acid insoluble fractions were screened using an open window for 10 minutes.
[0233]
[0234] Conversion with bisulfite, cloning and sequencing
[0235]
[0236] The bisulfite conversion was carried out according to the user guide of the MetilEasy Xceed kit (Human Genetic Signatures, cat. No. ME002). Cloning was performed using a TOPO TUn kit (Invitrogen, cat. No. K4595-40). Sequencing was carried out by applying the procedure described by Sanger.
[0237]
[0238] 5hmC dilution test principle test
[0239]
[0240] A 112 bp cDNA oligo containing three CpG sites was used in which one was hemi-5meC, the second CpG contained no modification and the third CpG was hemi-5hmC, as proof of the 5hmC dilution test principle. The oligo (65 ng) was added to a mixture of 5.0 jl 10x DNMT1-buffer (NEB), 2.5 jl of 3.2 mM SAM, 0.5 jl BSA (NEB, cat. No. B9001S) and 10 units of mouse DNMT1 in a total volume of 50 jl adjusted with MqH2O. DNMT1 reactions were incubated in a thermomixer at 37 ° C, 600 rpm for 4 h. Then, the DNA oligos with a MinElute Reaction Cleanup Kit. Bisulfite conversion and sequencing of the unmodified lower chain of the oligo was carried out before and after treatment with DNMT1.
[0241]
[0242] 5hmC dilution test
[0243]
[0244] A 112 bp cDNA oligo containing three CpG sites was used, one that had 5meC in both chains, a second that had no modification and a third that had 5hmC in both chains to demonstrate the 5hmC dilution test. To prepare hemi-modified oligonucleotides, the PCR was adjusted and started as follows: The oligonucleotide (65 ng) was added to a mixture of 4.0 pl of 5x Phusion HF-buffer, 1.6 pl of 2, 5 mM dNTPs, 1 pl of each of the 10 pM direct and reverse primers, 0.2 pl of Phusion polymerase in a total volume of 20 pl adjusted with MqH2O. Fusion of the DNA chains was carried out for 3 min at 98 ° C, followed by hybridization of the primer for 2 min at 56 ° C and elongation for 8 min at 72 ° C. Next, the DNA was purified with a MinElute Reaction Cleanup kit, the concentration was measured by fluorometry in a Qubit instrument and the DNMT1 treatment was carried out according to the following protocol: the total amount of oligo recovered was added to a mixture of 5.0 pl 10x DNMT1-buffer (NEB), 2.5 pl of 3.2 mM SAM, 0.5 pl BSA (NEB, cat. no. B9001S) and 10 units of mouse DNMT1 in a total volume 50 pl adjusted with MqH2O. DNMT1 reactions were incubated in a thermomixer at 37 ° C, 600 rpm for 4 h. Next, 1 pl of Proteinase K (14-22 mg / ml) (Roche) was added and incubation was continued at 50 ° C in a thermomixer, 600 rpm for 1 h. Then, the DNA oligos were precipitated with ethanol and further purified with a MinElute Reaction Cleanup Kit. The DNA concentration was measured again by fluorometry in a Qubit instrument. The protocol described in this section can be carried out one or more times to result in a dilution interval of 5hmC and conservation of 5meC.
[0245]
[0246] 5hmC dilution / loss test that allows specific chain evaluation
[0247]
[0248] A 112 bp ADNbc oligo cDNA containing three CpG sites was used, one that had 5meC in both chains, one second that had no modification and a third that had 5hmC in both chains to demonstrate the 5hmC dilution test (also called the assay of loss of 5hmC and primer extension assay ((biotin)) using the specific chain evaluation. To prepare hemi-modified oligonucleotides, the chain-specific primer extension PCR was adjusted and started as follows: the oligonucleotide (65 ng) was added to a mixture of 4.0 pl of 5x Phusion HF-buffer, 1.6 pl of 2.5 mM dNTPs, 1 pl of only one between the direct and reverse 10 pM primers containing a 5 'biotin molecule / label, 0.2 pl of Phusion polymerase in a total volume of 20 pl adjusted with MqH2O. Melting of the DNA chains was carried out for 3 min at 98 ° C, followed by hybridization of the primer for 2 min at 56 ° C and elongation for 8 min at 72 ° C. Next, the DNA was purified with magnetic beads coated with streptavidin and treatment with DNMT1 was carried out according to the following protocol: the total amount of oligo recovered was added to a mixture of 5.0 pl 10x DNMT1-buffer ( NEB), 2.5 pl of 3.2 mM SAM, 0.5 ml BSA (NEB, cat. No. B9001S) and 10 units of mouse DNMT1 in a total volume of 50 pl adjusted with MqH2O. DNMT1 reactions were incubated in a thermomixer at 37 ° C, 600 rpm for 4 h. Next, the biotinylated oligonucleotides were purified using streptavidin magnetic beads and converted with bisulfate, used as matrices in the PCR and sequenced.
[0249]
[0250] Results
[0251]
[0252] An outline of the procedure is depicted in Figure 1. To demonstrate the feasibility and success of the procedure, it will be tried to demonstrate that (i) specific methyl transferases preferentially modify hemi-5meC DNA substrates, (ii) that this preference can be identified by bisulfite sequencing after treatment with the appropriate methyl transferase and (iii) the 5hmC modification can be diluted with successive rounds of DNA amplification followed by treatment with the appropriate DNA methylase.
[0253]
[0254] Mouse DNMT1, human DNMT1 and M. SssI methyl transferase preferably hemi-5meC substrate methylate
[0255]
[0256] Mouse DNMT1, human DNMT1 and M. SssI methyl transferase were incubated with 100 ng unmodified, hemi-5meC, hemi-5hmC or hemi-beta-glucosyl-5hmC. Mouse DNMT1 was able to catalyze the transfer of a methyl group exclusively for the hemi-5meC substrate, presenting no activity on the other substrates (Figure 3A). Human DNMT1 presented an enzymatic preference for hemi-5meC while presenting a limited activity in the other substrates (Figure 3B). Finally, M. SssI methylase (Spiroplasma sp.) Also presented a preference for DNA containing hemi-5meC; (Figure 3C). From this result it was concluded that any of these methyl transferases could be sufficient for the dilution test described in Figure 1.
[0257]
[0258] Mouse DNMT1 has a solid preference for hemi-5meC as a substrate
[0259]
[0260] A cDNA substrate containing a hemi-5meC, unmodified cytosine and hemi-5hmC was incubated with mouse DNMT1 in the presence of S-adenosyl methylmethionine. The DNA was cleaned and subjected to bisulfite sequencing as described in "Materials and Procedures". After bisulfite sequencing, it was possible to demonstrate that virtually all (87.5%) hemi-5meC was methylated while unmodified CpG and hemi-5hmC remained unmodified with mouse DNMT1 (Figure 4). Since the substrate used for this assay simulates total 5hmC or total 5hmC DNA after a round of amplification, it was determined that the assay could work using it with multiple rounds of DNA amplification and treatment with mouse DNMT1.
[0261]
[0262] Successive rounds of treatment with DNMT1 and PCR amplification can dilute 5hmC while maintaining 5meC
[0263]
[0264] A cDNA substrate containing 5meC complete, CpG and 5hmC complete was amplified using Taq or Phusion polymerase followed by treatment with mouse DNMT1. This procedure was carried out three times, as described in "materials and procedures". Figure 6B demonstrates the effective dilution of 5hmC while maintaining 5meC. It can be seen that before dilution (Figure 6A) the identity of 5hmC and 5meC cannot be distinguished; however, after dilution treatment (Figure 6B); 5hmC and 5meC can be clearly distinguished since 5hmC is present in a greatly reduced amount compared to 5meC.
[0265]
[0266] Chain-specific primer extension PCR combined with the use of biotinylated primers allows evaluation of the DNMT1 transfer rate of methyl groups at CpG sites in a newly synthesized chain at sites transversely from 5hmC, 5meC or C.
[0267]
[0268] A cDNA substrate containing 5meC complete, CpG and -5hmC complete was used as a matrix for the primer-specific primer extension PCR with primers containing a 5 'biotin label and followed by treatment with mouse DNMT1. The newly synthesized oligonucleotides were isolated to ensure that no methylation / signal at the three CpG analysis sites of the chain selected for the study came from the parental copy of the same chain. This strategy allows direct detection and quantification of the level of 5meC and 5hmC without subsequent rounds of testing. The same assay that contains the oligonucleotide described in the heading materials and procedures can be used as internal reference and control to help calculate the content of modified C bases in genomic DNA samples.
[0269]
[0270] A representative protocol for the methyl transferase-dependent assay (test "B" in Figure 11) is as follows:
[0271]
[0272] Primer-Biotin extension: "One round" PCR w / biotinylated primer
[0273] Clustered PCR products (from the oligo control genomic sample)
[0274] MinElute PCR Purification
[0275] Biotin-streptavidin purification with MyOne ™ Streptavidin T1 beads
[0276] Treatment with DNMT1 on pearls, 0.6-1 pl of 0.5 mg / ml DNMT1, 1.6 mM SAM, 37 ° C in 30 min.
[0277] Washes; and elution in 50 pl MQ-H2O 95 ° C in 10 min.
[0278] MinElute Reaction Cleanup (optional)
[0279] Bisulfite treatment
[0280] Bisulfite PCR
[0281] TOPO TA cloning, transformation, selection on plates LB amp X-gal
[0282] Sequencing
[0283]
[0284] As shown in Figure 12, the HyLo methyl transferase / DNMT1-dependent assay identified two 5hmC CpGs in the gene TRIM31 gene in human brain DNA. The assay outlined in Figure 11 with genomic DNA with additions with a control oligo containing known CpG sites for each 5meC, C and 5hmC was used. According to this, an accurate quantification of genomic DNA could be ensured, since the use of oligo for the efficiency of methyl transferase within the sample was monitored.
[0285]
[0286] Example 2
[0287]
[0288] The addition of a chemical group can be carried out at 5hmC, such as glucose to improve the efficiency ratio of methyl transferase between 5meC and 5hmC. See Figure 13. Steric methyl transferase blockade in the modified 5hmC position can take advantage of the increased solidity of the methyl transferase-dependent assay. This paper demonstrates the blocking effect of glucose addition for 5hmC in a radioactive methyl transferase assay. It is possible to block both DNMT1 and M.Sssl by adding a chemical group with a larger size than that which can fit in a pocket of methyl transferase, for example, by adding glucose. As can be deduced from the data and the previous demonstration that the cytosine carbon-5 group of -CCCH3 (size 6.1 A) does not fit in the pocket of methyl transferase (Valinkluck and Sovers, Cancer Res, 2007) it can be assumed that the addition of any chemical group at 5hmC that makes the total group too large for the methyl transferase pocket is useful for increasing the efficiency rate of methyl transferase between 5meC and 5hmC.
[0289]
[0290] REFERENCES
[0291]
[0292] 1. Penn, N.W. Modification of brain deoxyribonucleic acid base content with maturation in normal and malnourished rats. Biochem J 155, 709-712 (1976).
[0293] 2. Cannon-Carlson, S.V., Gokhale, H. & Teebor, G.W. Purification and characterization of 5-hydroxymethyluracil-DNA glycosylase from calf thymus. Its possible role in the maintenance of methylated cytosine residues. J Biol Chem 264, 13306-13312 (1989).
[0294] 3. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science (New York, N. Y 324, 930-935 (2009).
[0295] 4. Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and

权利要求:
Claims (14)
[1]
1. Method for detecting 5-methylated and 5-hydroxymethylated cytosine residues in a nucleic acid sample comprising:
a) replication of said nucleic acid sample under conditions such that the 5-methylated cytosine residues are maintained and said hydroxymethylated cytosine residues are diluted;
b) treating said replicated nucleic acid sample to convert cytosine residues without modifying uracil or thymidine residues;
wherein said nucleic acid sample is divided into at least a first and a second portion and said replication and treatment steps are performed in said first portion, and the comparison of the sequence of said first nucleic acid sequence with the sequence of said second nucleic acid portion, wherein said hydroxymethylated cytosine moieties are identified as moieties that are read by sequencing as uracil or thymidine moieties in said first nucleic acid moiety and as cytosine moieties in the corresponding position in said second portion of nucleic acid and in which 5-methylated cytosine moieties are identified as moieties that are read as cytosine moieties in both mentioned portions of first and second nucleic acid.
[2]
2. The method of claim 1, wherein said treatment of said first and second portions to convert unmodified cytosine residues into thymidine residues further comprises treating said portions of first and second nucleic acid with bisulfite to convert cytosine residues without modifying uracil residues and replicating said first and second nucleic acid portions with a polymerase to convert said uracil residues to thymidine residues.
[3]
3. The method of claim 1, wherein said replication of said first portion further comprises:
a1) replication of said nucleic acid with a labeled primer to provide labeled replicated nucleic acid;
a2) treatment of said replicated nucleic acid chains labeled with a methyl transferase DNA to provide modified nucleic acid modified with labeled 5-methylcytosine;
a3) isolation of said modified nucleic acid modified with labeled 5-methylcytosine;
a4) treatment of said replicated nucleic acid modified with bisulfite-labeled labeled 5-methylcytosine to convert unmodified cytosine residues into uracil residues; Y
a5) replication of said labeled bisulfite treated nucleic acid isolated with a polymerase to provide a first portion of bisulfite treated nucleic acid.
[4]
4. The method of any one of claims 1 to 3, wherein said hydroxymethylated cytosine moieties are selected from the group consisting of 5-hydroxymethyl cytosine and b-glu-5-hydroxymethyl cytosine.
[5]
5. The method of claim 1 for detecting methylated and hydroxymethylated cytosine residues in a nucleic acid sample comprising:
a) dividing said sample into at least a first and second untreated portions;
b) replication of said first portion with a labeled primer and a polymerase to provide labeled parental and replicated nucleic acid;
c) treating said parental and replicated nucleic acid chains labeled with a methyl transferase DNA to provide modified nucleic acid modified with labeled 5-methylcytosine;
d) isolating said modified nucleic acid modified with labeled 5-methylcytosine;
e) treatment of said replicated nucleic acid modified with bisulfite-labeled labeled 5-methylcytosine to convert unmodified cytosine residues into uracil residues;
f) replication of said labeled bisulfite treated nucleic acid isolated with a polymerase to provide a first portion of bisulfite treated nucleic acid;
g) sequencing said first portion of bisulfite treated nucleic acid;
h) treatment of said second portion with bisulfite to convert unmodified cytosine residues into uracil residues;
i) replicating said bisulfite treated nucleic acid with a polymerase to provide a second portion of bisulfite treated nucleic acid;
j) sequencing said second portion of bisulfite treated nucleic acid; Y
k) comparison of the sequence of said first bisulfite treated nucleic acid with the sequence of said second bisulfite treated portion, wherein the 5-hydroxymethylated cytosine residues are identified as residues that are read by sequencing as a residue of uracil or thymidine in said first portion of bisulfite treated nucleic acid and as a cytosine moiety in the corresponding position in said second portion of bisulfite treated nucleic acid and in which the 5-methylated cytosine moieties are identified as moieties that are read as cytosine residues in said first and second portions treated with bisulfite.
[6]
6. The method of claim 4 or 5, wherein said labeled primer is a biotinylated primer.
[7]
7. The method of any one of claims 1 to 6, wherein said replication in step a) of the The method of claim 1 or the replication in steps b), f) and i) of the method of claim 5 is by a polymerase chain reaction, preferably a primer extension reaction, said replication preferably using a biotinylated primer.
[8]
8. The method of any of the preceding claims, wherein:
said replication steps a) and treatment b) in the method of claim 1 are carried out one or more times, said steps being preferably repeated 5 times or more, 7 times or more, 10 times or more or from about 1 to about 20 times or more or
said steps b), c) and d) in the process of any of claim 5 are repeated from about 2 to about 20 times, and / or
wherein said stages e and h are repeated from about 2 to about 20 times.
[9]
9. The method of any of the preceding claims, wherein, in the method of claim 1, the conditions in the replication step a), wherein said 5-methylated cytosine moieties are maintained and the moiety residues are diluted 5-hydroxymethylated cytosine, comprises the replication of said nucleic acid with a polymerase to provide replicated nucleic acid and the treatment of said nucleic acid replicated with an enzyme to 5-methylate the cytosine residues, said enzyme being preferably a methyl transferase DNA, selecting The methyl transferase DNA in the process preferably from the group consisting of mouse DNMT1, human DNMT1 and M.SssI DNMT.
[10]
10. The method of any of the preceding claims, further comprising the step of modifying 5-hydroxymethylated cytosine moieties in said samples to prevent methylation of said 5-hydroxymethylated cytosine moieties, said 5-hydroxymethylated cytosine moieties preferably being modified by the addition of a blocking group, said blocking group being preferably selected from the group consisting of beta-glucose, alpha-glucose, 6-OpD-glucopyranosyl-D-glucose, 6-O-alpha-D-glucopyranosyl-D-glucose; keto-glucose; azide-glucose; and modified versions thereof.
[11]
11. The method of any of the preceding claims, wherein said nucleic acid sample is selected from the group consisting of samples of human, plant, mouse, rabbit, hamster, primate, fish, bird, cow, sheep , pig, viral, bacterial and fungal; and or wherein said nucleic acid sample is genomic DNA.
[12]
12. The method of any of the preceding claims, further comprising comparing the presence of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid in said sample with a reference standard, in which an increase or decrease in the level of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid is indicative of the presence of a disease or of the probable course of a disease, and / or further comprising the step of providing a diagnosis or prognosis of a disease based on an increase or decrease in the level of 5-hydroxymethylcytosine and / or 5-methylcytosine in said nucleic acid compared to a reference standard, said disease being preferably cancer.
[13]
13. Procedure for predicting a predisposition to a disease in a subject, diagnosing a disease in a subject, predicting the likelihood of disease recurrence in a subject, providing a prognosis for a subject with a disease or selecting a subject with a disease for treatment with a particular therapy, which includes:
a) providing a genomic DNA sample of said subject; Y
b) detecting the state of methylation and hydroxymethylation of predetermined portions of said genomic DNA sample by the method of any one of claims 1 to 13,
wherein an alteration of the level of methylation of 5-hydroxymethylcytosine and / or 5-methylcytosine of said predetermined portions of said genomic DNA with respect to a state of reference methylation and / or hydroxymethylation provides an indication selected from the group consisting of an indication of a predisposition of the subject to a disease, an indication that the subject has a disease, an indication of the probability of recurrence of a disease in the subject, an indication of the subject's survival and an indication that the subject is a candidate for treatment with a particular therapy, said disease being preferably a cancer, said subject being preferably a human being.
[14]
14. A kit for determining the state of methylation and hydroxymethylation of a sample of nucleic acid comprising:
1) container (s) with reagents for methylation of nucleic acid; Y
2) container (s) with reagents for bisulfite sequencing;
3) a computer-readable medium comprising a computer program product that analyzes the sequence data obtained using said kit according to the procedure of claims 1-13; and said kit further comprising preferably nucleic acid primers for amplification and / or sequencing of a region of said nucleic acid sample.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201161570066P| true| 2011-12-13|2011-12-13|

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US201161570066P|2011-12-13|

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PCT/US2012/069525|WO2013090588A1|2011-12-13|2012-12-13|Methods and kits for detection of methylation status|

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