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    • 专利摘要:
    METHOD FOR THE INSULATION OF NUCLEIC ACIDS AND ITS KIT. The present disclosure provides a method for isolating artificial and natural nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and peptide nucleic acid (PNA) from a solid or liquid sample using cotton. Packaged cotton is such that a solution containing nucleic acids passes through the cotton and the nucleic acids in the solution are bound to the cotton in an ideal bonding medium. The nucleic acids bind to cotton so that the bound nucleic acids can withstand multiple washes with a liquid comprising water and elute in an aqueous buffer, with which the eluted nucleic acids can be directly used for amplification using PCR or for any other biochemical or molecular biological needs.
    公开号:BR112012016774B1
    申请号:R112012016774-0
    申请日:2011-01-06
    公开日:2020-12-01
    发明作者:Kumar Pullela Phani；Narayanan Manoj Malakkapurath；Kumar Gandhavalia Santhosh；Preetham Pinto Mitchell；Braskaran Chandrasekhar Nair；Venkata Pillarisetti Subbarao
    申请人:Bigtec Private Limited；
    IPC主号:
    专利说明:

    The present disclosure relates to the isolation and purification of nucleic acids. More essentially, it provides a method and kit for isolating and purifying nucleic acids using cotton or its derivatives. BACKGROUND AND BACKGROUND TECHNIQUE OF REVELATION
    Protocols for nucleic acid extraction can be broadly classified into silica-based and non-silica-based protocols. Existing silica-based and non-silica-based protocols cannot tolerate a water wash to remove non-nucleic acid components and require an aqueous wash with some percentage of alcohol present. The presence of alcohol in the eluted nucleic acid solution inhibits the polymerase chain reaction (PCR) and thus, normally both protocols require high-speed rotation or other methods for removing residual alcohol and eluting nucleic acids with a buffer aqueous at room temperature or elevated temperature. In certain cases, both protocols require a high salt concentration with polyethylene glycol or a wash with aqueous alcohol. The use of high concentration of salts and aqueous alcohol places a restriction on the elution of nucleic acids such as the complete removal of these components before the nucleic acids are eluted or the use of centrifuges, etc. Thus, none of the existing silica-based or non-silica-based protocols can be used non-POINT OF CARE (POC) as a centrifuge generates aerosols. Some of the non-silica based protocols reported in the literature are provided below:
    US patent 7264927: This document describes the use of cellulose or cellulose paper involving the use of polyalkylene glycol and high salt concentrations for binding and finally, the elution of nucleic acids in a buffer or in deionized water.
    United States Patent 6084091: Describes the method of using cellulosic flour (such as potato starch) for the isolation of nucleic acids.
    United States patent 5804684: Describes the method of using filter paper for the extraction of nucleic acid, where it is housed in a material such as a plastic tip with the help of soft tissue paper or a cotton piece as a filter or barrier to support the filter paper.
    All of the above processes use non-commercially available silica columns for the isolation of the final nucleic acid, or require longer processing times for samples (greater than 30 mins) or involve the use of high concentrations of salts during matrix washing or use of centrifuges, etc. None of the cellulose-based nucleic acid extraction methods washes the nucleic acids with a 100% aqueous buffer or water and usually contains a percentage of compounds containing polyol or alcohols. DECLARATION OF THE REVELATION
    Thus, the present disclosure reports a method for isolating nucleic acid from a sample, said method comprising the steps of: (a) adding a lysed buffer to the sample containing nucleic acid to obtain a lysed solution, or (b ) adding a lysed buffer in combination with a sample binding buffer to obtain a lysed solution, (c) adding a binding buffer to the solution from step (a) to bind the nucleic acid to a matrix or direct binding of the solution step (b) to a matrix, and (d) washing and eluting the nucleic acid bound to the matrix to isolate and purify the nucleic acid; and a kit for isolating nucleic acid from a sample, said kit comprising a matrix and buffers. BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
    The characteristics of the present disclosure will become more fully apparent from the following description and the attached claims, taken in conjunction with the accompanying figures. The understanding that these figures only show various achievements according to the disclosure, reveals that, therefore, they should not be considered as limiting the scope, as the disclosure will be described with additional details and specificities with the use of the accompanying figures:
    Figure 1: Cotton wrapped in [a] syringe [b] syringe needle [c] plastic molds attached to the syringe [d] plastic bottle with screw cap [e] glass test tube [f] glass bottle with cap screw thread.
    Figure 2: Cotton packed in [a] disposable plastic container [b] Pasteur molded plastic pipette [c] Pasteur glass pipette [d] plastic container with rubber trigger [e] cotton swab [f] molded plastic pipette.
    Figure 3: Cotton packed in [a] Eppendorf Tube [b] glass tube with screw cap [c] 1mL molded plastic tip [d] disposable graduated glass pipette with rubber driver [e] viscose swab [f] glass pipette with plastic and rubber actuator.
    Figure 4: DNA samples purified by different protocols were amplified by PCR. Field 1: Molecular weight marker, field 2: viscose packed in a 1 mL pipette tip, field 3: commercial viscose swab, field 4: cotton packed in 1 mL pipette tip, field 5: commercial silica column , field 6: DNA purified using commercial cotton swab, field 7: non-amplified DNA, field 8: water white.
    Figure 5: DNA samples purified by different protocols were amplified by PCR. Field 1: cotton packed in 1 mL pipette tip, field 2: water white, field 3: cotton packed in 2 mL syringe, Field 4: commercial silica column, field 5: molecular weight marker.
    Figure 6: DNA samples purified by different protocols were amplified by PCR. Field 1: molecular weight marker, Field 2: commercial silica protocol, Field 3: Cotton packed in 1 mL pipette tip, Field 4: Whatman paper filter No 1 packed in a pipette tip, Field 5: FTA card.
    Figure 7: A 30 ct RNA sample purified by different protocols was amplified by RT-PCR. Field 1: Molecular weight marker, Field 2: Surgical cotton, field 3: Autoclaved cotton, field 4: cotton washed with sodium hydroxide Field 5: cotton washed with hydrochloric acid, field 6: Absorbent cotton, Field 7: Qiagen silica column , Field 8: FTA card
    Figure 8: A cartridge component packaged in cotton for automated nucleic acid extraction. DETAILED DESCRIPTION OF THE REVELATION
    In the following detailed description, reference is made to the accompanying drawings, of which they form part. The illustrative achievements described in the detailed description, drawings and claims are not limiting. Other achievements can be used, and other changes can be made, without abandoning the spirit or scope of the subject presented here. It will be readily understood that aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be combined in a wide variety, all of which are explicitly contemplated and form part of this disclosure.
    The present disclosure relates to a method for isolating nucleic acid from a sample, said method comprising the steps of: (a) adding a lysed buffer to the sample containing nucleic acid to obtain a lysed solution; or (b) adding a lysed buffer in combination with a sample binding buffer to obtain a lysed solution; (c) adding a binding buffer to the solution of step (a) to bind the nucleic acid to a matrix or directly binding the solution of step (b) to the matrix; and (d) Wash and elute the nucleic acid bound to the matrix to isolate and purify the nucleic acid.
    In one embodiment of the present disclosure, the nucleic acid is selected from a group comprising DNA, RNA and PNA.
    In another embodiment of the present disclosure, the sample is a biological or non-biological sample.
    In yet another embodiment of the present disclosure, the biological sample that is selected from a group comprising blood, phlegm, serum, saliva or tissue extracts and the non-biological sample is selected from a group comprising chemically synthesized PNA.
    In yet another embodiment of the present disclosure, the lysed buffer is selected from a group comprising guanidine thiocyanate, guanidine hydrochloride, EDTA, Tris, detergent, polyol, monovalent salt containing group IA cation or divalent salt containing group IIA and protein digesting enzyme optionally along with urea or any of its combinations.
    In yet another embodiment of the present disclosure, EDTA has a concentration ranging from about 10 mM to about 300 mM, preferably about 100 mM.
    In yet another embodiment of the present disclosure, guanidine thiocyanate or guanidine hydrochloride has a concentration ranging from about 0.1 M to about 7 M.
    In yet another embodiment of the present disclosure, urea has a concentration ranging from about 0.01 M to about 7 M.
    In yet another embodiment of the present disclosure, Tris has a concentration ranging from about 0.01 mM to about 100 mM, preferably about 20 mM.
    In yet another embodiment of the present disclosure, the polyol has a concentration ranging from about 0.01% to about 30% (v / v).
    In yet another embodiment of the present disclosure, the detergent is selected from a group comprising sodium lauryl sulfate, sodium dodecyl sulfate, Triton X-100, Tween 20 and NP-40 or any combination thereof and in which the protein digesting enzyme is proteinase K.
    In yet another embodiment of the present disclosure, a binding buffer is water optionally together with polyols or non-polyols.
    In yet another embodiment of the present disclosure, the polyol comprises water-soluble polyol compounds selected from the group consisting of polyethylene glycol, glycerol, polypropylene glycol, ethylene glycol and propylene glycol.
    In yet another embodiment of the present disclosure, the non-polyol comprises alcohols consisting of methanol, ethanol, propanol or any water-soluble liquid with a functional group of acid, amine, alcohol, phenol, amide or ester as one of the functional groups; or any of its combinations.
    In yet another embodiment of the present disclosure, washing and elution are performed using wash buffer and elution buffer, respectively.
    In yet another embodiment of the present disclosure, the wash comprises a first wash with a wash buffer comprising about 1% to about 99% (v / v), preferably about 30% to about 70% (v / v) and ideally about 50% (v / v) aqueous alcohol followed by multiple washes with a wash buffer comprising 100% water.
    In yet another embodiment of the present disclosure, aqueous alcohol is selected from the group comprising ethanol, methanol, n-propanol, 2-propanol, glycerol, PEG, PPG, ethylene glycol and propylene glycol.
    In yet another embodiment of the present disclosure, water is selected from a group comprising deionized water, DNase-free water, RNase-free water, MilliQ water, filtered water, tap water and soil water or any combination thereof.
    In yet another embodiment of the present disclosure, said wash buffer can optionally comprise salts selected from a group comprising MgCl2, CaCl2 NaCl and KCl, or buffers selected from a group comprising bicin, tricine, Tris, HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bistris, PIPES, ACES, BES, TES, borate, TAPS, CHES, CAPS, ethanolamine and piperidine, with a pH ranging from about 5 to about 12.
    In yet another embodiment of the present disclosure, the elution buffer comprises hot water with a temperature ranging from about 45 ° C to about 99 ° C together with buffer or salt, with a pH ranging from about 8 to about 11.
    In yet another embodiment of the present disclosure, water is selected from a group comprising deionized water, DNase-free water, RNase-free water, MilliQ water, filtered water, tap water and soil water or any combination thereof.
    In yet another embodiment of the present disclosure, the buffer is selected from a group comprising bicin, tricine, Tris, HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bis-tris, PIPES, ACES, BES, TES, borate , TAPS, CHES, CAPS, ethanolamine and piperidine or any of their combinations with pH ranging from about 5 to about 12 or having pKa ranging from about 7 to about 10, In yet another embodiment of the present disclosure, salt is selected from a group comprising MgCI2, CaCI2, NaCI and KCI or any combination thereof in concentration ranging from about 0.01 mM to about 100 mM, preferably in the range of about 5 mM to about 50 mM.
    In yet another embodiment of the present disclosure, the matrix is selected from a group comprising cotton, cotton derivatives and synthetic polymers with cotton blends or any combination thereof.
    In yet another embodiment of the present disclosure, cotton is selected from a group comprising natural cotton, surgical cotton, clinical cotton, commercial cotton, woven cotton, water washed cotton, acid or base washed cotton, autoclaved cotton, cotton treated with buffer with pH ranging from about 1 to about 14, cotton treated with saline, cotton treated with organic solvent, pressed cotton and processed cotton.
    The present disclosure further relates to a kit for isolating nucleic acid from a sample, said kit comprising a matrix and buffers.
    In one embodiment of the present disclosure, the matrix is selected from a group comprising cotton, cotton derivatives and synthetic polymers with cotton blends or any combination thereof.
    In another embodiment of the present disclosure, cotton is selected from a group comprising natural cotton, surgical cotton, clinical cotton, commercial cotton, woven cotton, water washed cotton, acid or base washed cotton, autoclaved cotton, tampon treated cotton with pH ranging from about 1 to about 14, cotton treated with saline solution, cotton treated with organic solvent, pressed cotton and processed cotton.
    In yet another embodiment of the present disclosure, the buffer is selected from the group comprising the lysed buffer, binding buffer, wash buffer and elution buffer as described above.
    In yet another embodiment of the present disclosure, the sample comprises biological or non-biological samples.
    In yet another embodiment of the present disclosure, the biological sample is selected from a group comprising blood, phlegm, serum, saliva or tissue extracts and the non-biological sample is selected from a group comprising chemically synthesized PNA.
    The present disclosure relates to a method for building a system for extracting nucleic acid using cotton. The cotton is housed in such a way that all the solutions mentioned in the extraction of the nucleic acid interact with the cotton.
    In another embodiment, specifications for the various materials used in the present disclosure are provided below:
    Cotton, cotton derivatives, materials comprising cotton and cotton-like materials: Soft cotton is obtained as a cocoon around a cotton seed. For the extraction of nucleic acid, cotton is the preferred material as a matrix. The cotton forms can be natural cotton, surgical cotton, clinical cotton, commercial cotton, woven cotton, cotton washed with water, cotton washed with acid or base, autoclaved cotton, cotton treated with buffer (pH 1-14), cotton treated with saline solution, organic solvent treated cotton, pressed cotton and processed cotton, etc. are adequate. Any cotton fabric or different forms of cotton or synthetic polymers with a cotton blend or materials containing cotton can be used for the extraction of nucleic acid. Materials such as wool, silk, cashmere, etc., whose fibers behave similarly to cotton are also considered as part of this revelation. Cotton produced by organic farms or using insecticides and pesticides is also considered as part of this revelation. Cotton produced in different geographies will have a slightly different composition, structure, color and quality, and cotton grown in all regions of the world will be considered as part of this revelation. Any product of cotton origin or a material that uses cotton in its manufacture is considered part of this disclosure and can be used for the extraction of nucleic acid.
    Buffer Used: The lysed buffer contains a high concentration of EDTA to allow the binding of nucleic acids to cotton and also for handling different types of samples (blood, phlegm, serum, saliva, tissue extracts, etc.). Variation of constituent salts is possible and the use of EDTA is given as an example and should not be considered as a limit on disclosure. Typically, any negatively charged molecule in high concentration can repel nucleic acids in solution and extend the binding to cotton. The lysed buffer comprises guanidine thiocyanate or guanidine hydrochloride, EDTA, Tris, a detergent, and optionally with urea, a polyol, a monovalent salt containing group IA cation or divalent salt containing group IIA cation, and proteinase K or any enzyme protein digester. Guanidine thiocyanate or guanidine hydrochloride can have a concentration between 0.1 and 7 M. Guanidine thiocyanate can be replaced by guanidine hydrochloride or urea in some applications and its concentration can also vary between 0.1 and 7 M. Urea is used to denature proteins and complement the function of guanidine salts and can range from 0 to 7 M. Typically, most of the literature reporting blood lysis protocols contains EDTA in the 1-20 mM range. Our lysed buffer can contain significantly different amounts of EDTA, preferably in the range of 10-300 mM, more preferably in the range of 100 mM. The concentration of EDTA can be manipulated for other nucleic acids such as RNA and PNA, but in general, it has been determined that a higher concentration of EDTA helps to improve time cycles (Ct) and signal strengths in PCR and RT-PCR. The significantly different concentration of EDTA helps to retain the iron present in hemoglobin (for blood), prevents any DNase and RNase activity, and creates a highly negative atmosphere in which the nucleic acid can bind to cotton. The important aspect of this is that, this realization is the first example in which the binding of DNA to a matrix is done under basic conditions. Importantly, the binding pH of the solution has a significant effect on DNA / RNA binding to cotton, so a pH of around 8-10 is preferred for binding, but a pH of 7.1-12 can also be used. . Magnesium chloride is typically used in higher concentrations to deactivate RNase activity and thus, no DNA lysis protocol is absent or is used minimally (about 20 mM). Again, the use of EDTA also disables RNase and the use of MgCI2 is not essential, being optional for any application of nucleic acid. Tris is the choice of the lysis buffer, and we think that 0-100 mM can be used, and typically around 20 mM is ideal. It should be noted that, in our lysed buffer, the role of Tris is to assist in lysis and can be replaced by any suitable buffer. The use of non-binding polyol is to improve the solubility of the cleaved and denatured proteins. The percentage of polyol in a binding buffer can be 0-30% (v / v). In some applications, a separate binding buffer is not added and after lysis, the nucleic acids are directly bound to the matrix. All of these buffers were typically made in deionized water and for RNA applications, the water can be optionally treated DEPC and autoclaved. Lysis of proteinase K can be done before adding the aforementioned lysed buffer to blood, phlegm, saliva, semen, etc. and optionally can be done with lysed buffer. The proteinase K treatment was determined to be the effective non-lysed buffer mentioned and thus, this treatment can be done before adding the lysed buffer or can be done together with the lysed buffer for any type of liquid sample containing nucleic acids. The used non-lysed buffer detergent can be selected from the group comprising SLS (sodium lauryl sulfate), SDS, Triton X-100, Tween 20, or any other commonly used ionic, non-ionic detergent known in the art.
    Binding buffer: The binding buffer, which was added after lysis to initiate the binding of nucleic acids to cotton, was determined to be very flexible in terms of composition and pH. The binding buffer is so flexible that only the addition of water is sufficient to dilute the concentration of the non-lysed buffer salts, with a good binding of nucleic acids to cotton. The cotton can be placed to interact during lysis or after adding a binding buffer to extract the nucleic acids from the given sample. For practical purposes, a binding buffer composition can have any pH between 4 and 12, preferably in the range of 7-10. For complex samples such as blood, phlegm or saliva, a binding buffer can have a certain percentage of polyol compounds soluble in water such as PEG, glycerol, PPG, ethylene glycol, propylene glycol, etc. The molecular weight of PEG and PPG can vary between 200-200,000 and for all practical purposes it will range from 1000 to 20,000. The percentage of polyol compounds in the binding solution can be up to 50% (v / v), but for all practical applications, it will be 1-30% (v / v). The polyol compounds must guarantee the complete miscibility of the lysed components and if the proteinase K lysis is complete, the percentage of polyol can be reduced to 1%. Buffers known in the art include bicin, tricine, Tris, HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bis-tris, PIPES, ACES, BES, TES, borate, TAPS, CHES, CAPS, ethanolamine, piperidine, etc. in the pH range 5-12, preferably in the range 7-10 can be used for the preparation of a binding buffer and the presence of the buffer is not mandatory, but can be used depending on the type of sample, the volume of the sample , the temperature, the composition of the lysed buffer. If buffering salts are used, their compositions can range from 1-200 mM, preferably 1-100 mM, and more preferably 5-50 mM. The concentration described above is the concentration of the binding solution and after addition to the lysed buffer the concentration changes depending on the composition of the lysed buffer. Also, the pH defined above is for the pH of a binding buffer when it was made and after addition to the lysed buffer the pH of the mixed solution (lysed buffer + binding buffer) may change. Although alcohols such as methanol, ethanol, and propanol are not polyols, they can also be used in the preparation of the binding buffer. In general, any water-soluble liquid with a functional group of acid, amine, alcohol, phenol, amide, ester, etc. can be used as one of the functional groups.
    Wash Buffer: A wash buffer is a solution, which selectively washes the non-nucleic acid components of cotton. If the clinical sample is blood, after binding, the cotton will be brown in color and to remove the color it was determined that a percentage of ethanol not wash buffer (called wash buffer 1) helps. In particular, the first wash was done with a buffer or water containing 1-99% (v / v), preferably 30-70% (v / v), more preferably 50% (v / v) ethanol. If necessary, multiple washes can be done with aqueous ethanol to get rid of non-nucleic acid components, depending on the sample. Methanol, n-propanol, 2-propanol, glycerol, PEG, PPG, ethylene glycol, propylene glycol or any other water-soluble alcohol can replace ethanol in the washing solution. A wash buffer 2 can be used where a monovalent or divalent cation is present together with the wash buffer 1 as its composition. It is also possible that the wash buffer 1 and 2 may have the same composition and comprise water, an alcohol and a monovalent or divalent cation. The number of times that the cotton can be washed with wash buffers 1 & 2 can be 0-10 and ideally in the range of 1 - 3. Other washes will normally be with deionized water and the number of washes can be one or ten, preferably 2 to 5 and more preferably 3-5. The deionized water used in the washing step can be replaced with water free of DNase, RNase, or MilliQ water or filtered or tap water or soil water. We observed an initial wash with aqueous alcohol tends to remove most non-nucleic acid components, followed by multiple washes with water (100% water) to remove residual alcohol. Water washes ensure that the nucleic acids obtained are ready PCR inhibitors with or without minimal PCR. The wash buffers can optionally contain salts such as MgCl2, CaCl2, NaCl, KCI, or buffers such as bicin, tricine, Tris, HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bis-tris, PIPES, ACES, BES, TES, borate, TAPS, CHES, CAPS, ethanolamine, piperidine, etc. in the pH range 5-12. The buffer or salt or a combination thereof may be in a concentration of 1-1000 mM, preferably in the range of 20-200 mM and more preferably around 100 mM. Elution buffer: Any hot aqueous buffer solution (45-99 ° C) can elute nucleic acids from cotton. The elution pH was determined to be crucial, but preferably in the range of 8-11 and elution should be done at a temperature between 45-99 ° C for complete recovery of the nucleic acids. The non-buffered deionized water that performs the elution step can be replaced with water free of DNase, RNase or MilliQ water or filtered or tap water or soil water. Buffers known in the art include bicin, tricine, Tris, HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bis-tris, PIPES, ACES, BES, TES, borate, TAPS, CHES, CAPS, ethanolamine, piperidine, etc. in the pH range 5-12 can be used for elution, although the most preferred are with pKa in the range 7-10, Whenever cotton is used for eluting bound nucleic acids, the elution buffer must be hot and for practical considerations, in the range of 45-99 ° C. This is in stark contrast to most of the methods mentioned in the literature, in which elution is done in hot conditions using deionized water, but the nucleic acids attached to cotton cannot be completely eluted with hot water, the presence of a buffer or salt. The salt can be MgCl2, CaCl2, NaCl, KCl, etc. in a concentration of 0-100 mM, preferably in the range of 5-50 mM. The buffer can be selected from a group of buffers, namely, bicin, tricine, Tris, HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bis-tris, PIPES, ACES, BES, TES, borate, TAPS, CHES, CAPS, ethanolamine, piperidine.
    Nucleic acid recovery: With the current system, nucleic acid recovery depends on the lysed buffer, the binding buffer, the washing buffers and the elution buffer, as well as the combinations used. Depending on the combination, nucleic acid recovery can be comparable to any silica-based nucleic acid extraction system. It has also been observed that with low-titration samples, the efficiency of our cotton-based nucleic acid approach is sometimes better than silica.
    Cotton Quantity: The cotton quantity is dependent on the volume of the clinical sample and for samples in the range of 1-300 μl, it was determined to be adequate 5-30 mg of cotton. For sample volumes in the milliliter range, 50 mg or more of cotton may be essential. Not generally, 1 milligram to 10 grams of cotton is sufficient to extract nucleic acids from any clinical, environmental or field samples.
    Price of each test: As the cotton used in this protocol is the surgical cotton available not sold in any pharmacy or retailer (can be autoclaved or subjected to purification), the price of each nucleic acid extraction is minimal and one of the lowest reported until the present in the literature. Again, considering the elimination of the presence of accidental PCR inhibitors in the nucleic acid eluates, the simplicity and adaptability of using the POC, ease of automation, etc. make this approach superior, and the cost per extraction is probably an extra bonus.
    Component safety and solution disposal: The cotton-based nucleic acid extraction system uses most water-based plugs, with which leftovers can be safely and effectively disposed of by a non-subject technician. The cotton can be packed in a cartridge and all lysis, binding, and washing solutions can be kept in a non-cartridge for POC use, with which the worker or health analyst has no need to discard leftovers and the cartridge will be self-contained.
    Uses of extracted nucleic acids: The extracted nucleic acids using the cotton protocol described in this embodiment can be ready for use in a PCR or RT-PCR. Other applications of the described cotton protocol are for the recovery of nucleic acids from a clinical sample for archiving, storage, other biochemical and molecular biology uses, etc. The extracted nucleic acids can be used for any biochemical or molecular biological application, which a non-subject technician may occasionally encounter.
    Use of cotton in a form suitable for the extraction of nucleic acid: This accomplishment serves to extract nucleic acids in ‘PCR-ready form’ using cotton with minimum equipment requirement like centrifuge or other rotating equipment. The cotton can be packaged in any form suitable for the extraction of nucleic acid depending on the amount of the sample, the nature of the sample and the origin of the sample. Preferably, the nucleic acids were obtained in a solution or emulsion, which will be processed according to the described systems of lysis, ligation, washing and elution. Thus, cotton packaging is an important part of extracting nucleic acid and any nature in which cotton may come into contact with the solution containing nucleic acids is considered as part of this disclosure. Figures 1-3 illustrate some ways in which cotton can be packaged, but it should not be construed as limiting in any way. In simple terms, cotton is packaged in such a way that the solution containing nucleic acid comes into contact with cotton or cotton comes into contact with the liquid, being considered as part of this disclosure.
    In another embodiment, the cotton can be packaged in a modified 1 mL plastic pipette tip, modified 2 mL plastic pipette tip, 15 mL falcon tube, 50 mL falcon tube, 1.5 mL eppendorf tube, 2 ml eppendorf tube, 5 ml borosilicate glass test tube, 4 ml screw cap plastic bottle, 3 ml plastic Pasteur pipette, glass Pasteur pipette, glass Pasteur pipette with rubber bulb, Pasteur pipette plastic with rubber bulb, glass pipette with plastic mold and rubber as bulb, 2 mL glass vial with plastic cap, 10 mL autoclaved disposable plastic syringe, plastic mold attached to the 5 mL syringe, disposable unit attached to the 50 mL syringe, etc. Cotton can also be made in the form of a cotton swab and the swab can be machine-made or manually. The cotton swab shown is Fig: 3 [e] made of viscose and any polymer mixed with cotton (1 to 100%), or chemically or physically modified cotton (1 to 100%) is considered as part of this disclosure.
    Use of cotton to store a clinical sample: Cotton can be directly exposed to sampling and, being absorbent, cotton will stabilize and store the sample safely. The safe form is defined as a medium in which the nucleic acid content of the added sample is not significantly degraded. The binding can be reversibly, where the nucleic acids can be extracted using the nucleic acid extraction protocol described in this embodiment. The cotton can optionally be integrated with a stabilizer, which improves the stability of the sample and the constituents of the sample. Optionally, the cotton can be integrated with an enzyme or a chemical or the lysed buffer communicated in this protocol or the lysed buffer containing proteinase K, or proteinase K together with stabilizing buffer salts. Cotton can be treated with EDTA, treated with sodium azide, treated with base, treated with acid, treated with lysed buffer, treated with honey, treated with any antibacterial agent, treated with any antimicrobial agent, treated with any antiviral compound or treated with EDTA and sodium azide, antibacterials, antimicrobials, antivirals, anticoagulants, stabilizers of clinical samples known in the art, honey, or any of its combinations. The volume of the sample added to the cotton for storage can be any volume, but for all practical reasons, it can be 1 μl to 20 mL and the amount of cotton used can be any quantity and for practical reasons it can be 1 mg to 10 grams . When the sample is collected, it can be made of cotton impregnated with a lysed buffer, and is also considered as part of this development.
    Method for using a packaged cotton system for nucleic acid extraction: Cotton packaged in a device as exemplified in Figure 1-3 using the communicated nucleic acid extraction protocol described in this embodiment. The mechanism by which the cotton interacted with the lysis, binding, washing and elution system described in this embodiment can be heating, agitation, vortexing, rotation, constant movement, pipetting, or any other means by which a solid and liquid can interact. Essentially, the liquid-containing nucleic acid will come in contact with the cotton fibers.
    In another embodiment, the present method is one of the simplest and most flexible protocols for nucleic acid extraction reported in the literature. Almost all methods in the literature require a centrifuge to rotate the content, or a magnet to hold the magnetic particles in contact position or both, and the method communicated in this embodiment completely eliminates the need for a centrifuge or a magnet. Existing protocols have limits on sample volume or require multiple processing for larger sample volumes. This protocol can process virtually any number of samples (for practical purposes, 1 μl to 20 mL) almost at the same time using a simple disposable extraction system. The present method produces nucleic acids immediately ready for further characterization and downstream processing such as PCR, sequencing or blotting. The simplicity of this system makes it equally suitable for point of care (POC) or established laboratories, first of a kind reported in the literature.
    The cotton protocol described in this realization has outstanding characteristics such as the elimination of the use of the centrifuge, minimum user for user variation, efficiency comparable to the silica protocols, ease of use compared to any existing nucleic acid protocol, ability to process any quantity sample size, adequate recovery of nucleic acids, ability to collect low titration samples, ease of automation, suitable for both hospital environments and point of care establishments and high consistency in the recovery and quality of nucleic acids.
    In another embodiment, the method described in this disclosure employs fibrous materials such as cotton to extract nucleic acids from virtually any clinical or analytical sample of biological origin in a reverse transcriptase PCR or PCR (RT-PCR) or ready format for sequencing or blotting. The procedure comprises lysis, binding of nucleic acids to cotton, washing the cotton attached to the nucleic acid with aqueous solutions, and eluting the nucleic acids in a buffer with salt such as KCl. A typical buffer lysed in silica or non-silica protocols contains some tetra or dibasic ions like EDTA (chelating agents), which bind to blood iron. In this communicated protocol, a high concentration of EDTA (10-300 mM) is added to create an environment in which all nucleic acids selectively bind to cotton. Normally the pH of the lysed buffer is adjusted to 6 to allow binding to a matrix (silica or non-silica), where most proteins and other components have a neutral or positive charge, where the nucleic acid is still negatively charged and interacts with a matrix. In the current system, the binding pH must be basic (pH 8-11) and the excess of negatively charged EDTA itself (10- 300 mM) acts as a buffer and takes the pH around 8. Ours is the first protocol wherein the nucleic acids are lysed and can be linked to a matrix with basic pH. The binding buffer can be water, any aqueous buffer with a pH in the range of 3-11, or polyethylene glycol containing water (PEG, 1-30%) or glycerol (1-30%) or polypropylene glycol (PPG, 1-30% ) or ethylene glycol (1-50%) or propylene glycol (1-50%) or any water-soluble alcohol or any combination above. The binding buffer serves to ensure dilution of the salts of the lysed buffer, to expand the binding of the nucleic acids in an EDTA-rich atmosphere and to solubilize the lysed particles. The communicated protocol can tolerate a wide range of buffers with different pHs for binding, this is also the first time in the literature that the pH of a binding buffer or composition is so flexible. The longer sample processing times, the limit on the sample volume and the non-possibility of quantifying nucleic acids are disadvantages associated with FTA cards, which do not exist in the nucleic acid extraction protocol reported in this realization using cotton. Finally, all protocols reported in the literature for elution in an aqueous buffer or water at room temperature or occasionally at high temperature and our nucleic acid washing are with water at room temperature and elution at high temperature (50-99 ° C). Using the protocol and matrix defined in this embodiment, the deionized hot water will not elute all bound nucleic acids and the presence of a buffer or salt or a combination of these is a necessity. This is also a stark contrast to the nucleic acid extraction protocol in the literature, which can elute bound nucleic acids from the matrix in hot deionized water (both the silica and non-silica protocols allow elution in nucleic acids in hot water). The elution buffer can be anything between pH 8-10, indicating that the elution pH is flexible, with some salt concentration as KCI being preferred for efficient elution of the bound nucleic acids from cotton.
    In another embodiment, in the methods below, cotton and other fibrous materials based on cotton were used in the quantitative extraction of nucleic acids under special lysis, binding, washing and elution conditions, which are exclusive for the elution of nucleic acids from cotton. The present disclosure in one aspect provides a quick system for isolating nucleic acid from any environmental, clinical, bacterial, fungal and animal source using cotton. Samples can be cell lysates, body fluids, plants, tissues and bacterial cells and cell lysates. Cotton and viscose are fibrous materials obtained naturally and artificially respectively, which have been determined to be bound to nucleic acids under certain conditions. The process of nucleic acid binding and the selective retention of nucleic acids in cotton and the release of nucleic acids under specific elution conditions are exemplified by DNA and RNA.
    In another embodiment, in a typical DNA extraction from a clinical sample such as blood, the blood was lysed with a lysed buffer comprising guanidine thiocyanate, EDTA, a buffer such as Tris, a detergent such as triton X-100, and optionally with urea, a polyol, a group IA cation containing monovalent salt and / or a group IIA cation containing divalent salt, and enzymes for dividing proteins such as proteinase K. Guanidine thiocyanate can be between 0.1 to 7M of concentration. Guanidine thiocyanate can be replaced by guanidine hydrochloride in some applications and its concentration can also vary between 1 to 6 M. Urea is used to denature proteins and complements the function of guanidine salts and can vary between 0 and 7 M Typically most of the lysis protocols reported in the blood literature contain EDTA in the 20 mM range. Our lysed buffer could tolerate significantly greater amounts of EDTA in the 10-300 mM range, preferably around the 100 mM range for efficient binding of nucleic acid to cotton. The EDTA concentration can be manipulated for other nucleic acids such as RNA and PNA, but in general, a higher concentration of EDTA helped in improving cycle times (Ct) and signal intensities in PCR and RT-PCR. The significant higher concentration of EDTA helps to retain the iron present in hemoglobin (for blood), prevents any DNase activity and creates a highly negative atmosphere in which the nucleic acid can bind to cotton. The important thing is that the addition of a significantly higher concentration of EDTA to the buffer makes the pH of the buffer basic and as far as we know, this achievement is the first example in which the nucleic acid that binds to a matrix is done under basic conditions. Importantly, the binding pH of the solution has to be basic to allow the binding of the nucleic acid to cotton, since with a very acidic pH, there is a chance that EDTA will precipitate out of the lysed buffer and thus a pH above 8 for binding. Magnesium chloride is typically used in higher concentrations to disable RNase activity and thus could be used in the nucleic acid lysis protocol. Tris is the choice of the lysis buffer, and we found that 0-100 mM can be used and that typically around 20 mM is ideal. The use of polyol in lysis or a binding buffer serves to increase the activity of Proteinase K and to improve the solubility of the cleaved proteins. The percentage of polyol in the lysed buffer can be 0-30% (v / v). All these plugs were made in deionized water and for the application of RNA, the water can be treated with DEPC and autoclaved. Lysis of proteinase K can be done before adding the aforementioned lysed buffer for blood, phlegm, saliva, etc. and together with the lysed buffer for urine, sweat, etc. The proteinase K treatment was determined to be effective in the mentioned lysed buffer and thus, this treatment could be prior to the addition of the lysed buffer or could be together with the lysed buffer for any type of clinical sample containing nucleic acids.
    In another embodiment, the binding buffer, which was added after lysis to initiate the binding of nucleic acids to cotton, was found to be very flexible in terms of composition. A binding buffer is so flexible that only the addition of water is sufficient to dilute the concentration of salts in the lysed buffer, and the good binding of nucleic acids to cotton is also observed. The cotton can be made to interact during lysis or after adding the binding buffer. The composition of a binding buffer can be at any pH between 5 to 12, preferably in the range of 7-10. For complex samples such as blood, phlegm or saliva, a binding buffer can have a certain percentage of polyol compounds such as PEG , glycerol, PPG, ethylene glycol, propylene glycol etc. Binding solutions traditionally used (for silica-based nucleic acid systems) such as ethanol or aqueous ethanol have been determined to reduce the binding affinity of nucleic acids to cotton, that is, the presence of ethanol during the binding step has reduced efficiency of nucleic acid binding to cotton.
    In another embodiment, a wash buffer is a solution, which selectively washes the non-nucleic acid components of the cotton. If the clinical sample is blood, after binding, the cotton will be brown in color and to remove the color it was determined that a percentage of ethanol in the wash buffer (called wash buffer 1) helps. In particular, the first wash was done with a buffer or water containing 10-90%, preferably 30-70%, more preferably 50% ethanol. Methanol, n-propanol, isopropanol, glycerol, PEG, PPG, ethylene glycol, propylene glycol or any other water-soluble alcohol can replace ethanol in the wash solution. A wash buffer 2 can be used where a monovalent or divalent cation exists together with wash buffer 1 as its composition. It is also possible that the wash buffer 1 and 2 may have the same composition and comprise water, an alcohol and a monovalent or divalent cation. The number of times the cotton can be washed with wash buffers can be 0-10 and ideally in the range 1-3. Other washes will normally be with deionized water and the number of washes can be between one and ten, preferably 2 to 5 and more preferably 3-5. The deionized water used in the washing step can be replaced by DNase, RNase-free water or MilliQ water, filtered water or tap water or soil water.
    In another embodiment, elution of the cotton nucleic acids can be done with any aqueous buffer. The concentration of the buffer must be between 1 and 200 mM, preferably 5-50 mM, more preferably, 30-70 mM in the elution buffer. Buffers known in the art include bicin, tricine, Tris, HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bis-tris, PIPES, ACES, BES, TES, borate, TAPS, Ethanolamine, CHES, CAPS, ethanolamine, piperidine, etc., in the pH range 5-12, preferably in the range of 7-10, more preferably in the range of 8-10, operate to elute the nucleic acids from cotton in warm conditions. The elution of the cotton nucleic acid must be carried out at an elevated temperature between 50 and 100 ° C, preferably at 70-95 ° C, more preferably at around 85 ° C.
    In another embodiment, purified nucleic acids can be 10-100% pure, and will normally be ready for PCR. The purity of the nucleic acids depends on the ideal combination of lysis, binding, washing and the elution buffers and the purification matrix (cotton or cotton derivatives or materials mixed with cotton). We observed that, FTA cards or cellulose-bound particles, or cellulosic filter papers are not efficient under these conditions of buffer combinations, indicating that cotton is different from other forms of cellulose in relation to the interaction with nucleic acids. In related methods, the present disclosure provides a means to isolate nucleic acids from a sample containing nucleic acids using cotton or cotton derivatives or materials mixed with cotton as the solid matrix using the following general protocol. a) The sample containing nucleic acids was added to the lysed buffer. The lysed buffer comprises guanidine thiocyanate, EDTA, Tris, a detergent, and optionally urea, a polyol, a group IA cation containing monovalent salt and / or a group IIA cation containing divalent salt, and proteinase K. The acid sample nucleic acid and the lysed buffer were mixed and heated to 50-95 ° C for 1-20 min. b) A binding buffer was added to the above solution which could be water, a buffer with a pH between 4-11, or a solution containing a polyol. The volume of the binding buffer could be 0.1-10 times the volume of the lysed buffer. c) The solution above was made to interact with cotton preferably at room temperature for a few seconds to a few minutes. d) Then, the cotton was specifically washed (first wash) with a wash buffer comprising aqueous alcohol or just water. e) The cotton above was then washed with water or a buffer until the residual alcohol was removed from the cotton. f) The nucleic acids were eluted with a buffer comprising a salt such as KCl (Salts containing cations of Group IA or Group IIA) and / or bicin as a buffer and the eluted nucleic acids are normally ready for use in PCR or RT-PCR.
    The present disclosure is further elaborated with the help of the following table, which provides a comparison between the method used in the present disclosure and those used in the prior art. The table compares some of the important aspects regarding the various methods used for the characterization of methods used for the isolation of nucleic acids.


    Table 1: Comparative table of the important aspects involved in the isolation of nucleic acids.
    The technology of the present disclosure is further elaborated with the help of the following examples. However, the examples should not be understood as limiting the scope of the disclosure.
    General Methodology: The solution containing nucleic acid was placed in contact with cotton preferably at room temperature and the non-nucleic components were washed from the cotton using a series of washes comprising aqueous alcohol and water. The cotton nucleic acids were eluted using an aqueous buffer 10 comprising an elevated temperature salt. The eluted nucleic acids will be ready for further processing or for PCR. The following examples are provided with cotton wool packed in a 1 mL pipette tip normally used in research laboratories. But as the technician in the field can see, cotton can be packaged in any form where there is an opportunity for a liquid to come into contact with it. Essentially, anything with an input and output and between the cotton that can be packaged is considered to be part of that revelation.
    Example-1: DNA extraction from blood a) 50 μl of blood was added to 75 μl of lysed buffer (30 μl of 10 mg / ml proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 2 min. b) 150 μl of a binding buffer (water with 0.1 g / ml PEG 6000) was added to the above solution. c) A 1 mL plastic dropper packaged with 8 mg of cotton (cotton droppers, as shown in Figure 2 [a]) was placed to interact with the above solution. d) Afterwards, the cotton swab was washed with 2 ml of each wash buffer 1 (50% ethanol) and wash buffer 2 (50% ethanol containing 100 mM MgCl2). e) The cotton tip was washed with water (3 X 1mL). f) The nucleic acids were eluted in 100 µL of elution buffer (10 mM bicin, 10 mM KOI, pH 9.8) at 95 ° C.
    Example-2: DNA extraction from blood a) 100 μl of blood was added to 150 μl of lysed buffer (40 μl of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 2 min. b) 300 μl of a binding buffer (water with 0.1 g / ml PEG6000) was added to the above solution. c) A molded 1 mL pipette tip packed with 10 mg cotton (cotton tip, as shown in Figure 3 [c]) was placed to interact with the above solution. d) Then, the cotton tip was washed with 1 ml of wash buffer 1 (50% ethanol) and 2 ml of wash buffer 2 (50% ethanol containing 100 mM MgCl2). e) The cotton tip was washed with water (3 X 1mL). f) The nucleic acids were eluted in 200 µL of elution buffer (10 mM bicin, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-3: DNA extraction from blood a) 100 μl of malaria parasite (p. Falciparum) containing blood was added to 150 μl of lysed buffer (40 μl of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate , 100 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 2 min. b) 300 μl of a binding buffer (water with 0.1 g / ml PEG6000) was added to the above solution. c) A molded 1 mL pipette tip packed with 10 mg cotton (cotton tip, as shown in Figure 3 [c]) was placed to interact with the above solution. d) Then, the cotton tip was washed with 1 ml of wash buffer 1 (50% ethanol). e) The cotton tip was washed with 2 ml of wash buffer 2 (50% ethanol containing 100 mM MgCl2). f) The cotton tip was washed with water (3 X 1 mL). g) The nucleic acids were eluted in 200 µL of elution buffer (10 mM bicin, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-4: RNA extraction from blood a) 50 μl of positive Chikungunya blood was added to 75 μl of lysed buffer (30 μl of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris , 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 2 min. b) 150 μl and a binding buffer (water with 0.1 g / ml PEG6000) were added to the above solution. c) A 1 mL pipette tip packed with 10 mg cotton was placed to interact with the above solution. d) Then, the cotton tip was specifically washed with 1 ml of wash buffer 1 (50% ethanol containing 50 mM MgCl2). e) The cotton tip was washed with water (3X1 mL). f) The nucleic acids were eluted in 200 µL of elution buffer (10 mM bicin, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-5: DNA extraction from saliva a) 50 μl of saliva was added to 100 μl of lysed buffer (10 μl of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate, 200 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 2 min. b) 250 μl of a binding buffer (10% glycerol in water) was added to the solution above. c) A molded 1 mL pipette tip packed with cotton (cotton tip, as shown in Figure 3 [c]) was placed to interact with the solution above. d) Then, the cotton tip was specifically washed with 3 ml of wash buffer 1 (50% ethanol containing 200 mM MgCl2). e) The cotton tip was washed with water (3 X 1mL) by pipetting the liquid three times during each wash. f) The nucleic acids were eluted in 250 μl of elution buffer (10 mM bicin, 50 mM KCl, pH 9.8) at 95 ° C.
    Example-6: RNA extraction from blood a) 100 μl of positive Chikungunya blood was added to 150 μl of lysed buffer (40 μl of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 2 min. b) 300 μl of a binding buffer (water with 0.1 g / ml PEG 6000) was added to the above solution. c) A molded 1 mL pipette tip packed with 10 mg cotton (cotton tip, as shown in Figure 3 [c]) was placed to interact with the above solution. d) Then, the cotton tip was washed specifically with 1 ml wash buffer 1 (50% ethanol) and 2 ml wash buffer 2 (50% ethanol containing 50 mM MgCl2). e) The cotton tip was washed with water (2 X 1mL). f) Nucleic acids were eluted in 100 µL of elution buffer (10 mM bicin, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-7: Extraction of RN A from blood a) 50 μl of positive Chikungunya blood was added to 75 μl of lysed buffer (40 μl of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate, 80 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 55 ° C and left at that temperature for 3 min. Then, the solution was heated to 70 ° C for 2 min. b) 150 μl of a binding buffer (water with 0.1 g / ml PEG 8000) was added to the above solution. c) A 2.5 mL syringe packaged with 10 mg of cotton (cotton syringe, as shown in Figure 1 [a]) was placed to interact with the above solution. d) Then, the cotton tip was specifically washed with 1 ml of wash buffer 1 (50% ethanol) and 2 ml of wash buffer 2 (50% ethanol containing 50 mM MgC12). e) The cotton syringe was washed with water (3 X 1mL). f) Nucleic acids were eluted in 100 µL of elution buffer (10 mM bicin, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-8: Extraction of phlegm DNA a) 100 μl of phlegm were added to 150 μl of lysed buffer (40 μl of 10 mg / ml proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 5 min. Then, the solution was heated to 75 ° C for 2 min. b) 300 μl of a binding buffer (water with 0.1 g / ml PEG 6000) was added to the above solution. c) A 5 mL bellows pipette packed with 10 mg of cotton (cotton bellows, as shown in Figure 2 [b]) was placed to interact with the above solution. d) Then, the cotton tip was washed specifically with 1 ml wash buffer 1 (50% ethanol) and wash buffer 2 (50% ethanol containing 50 mM MgCb). e) The cotton bellows was washed with water (3 X 1mL). f) Nucleic acids were eluted in 100 µL elution buffer (10 mM tricine, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-9: DNA extraction from serum a) 50 μl of serum was added to 75 μl of lysed buffer (60 μl of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 2 min. b) 150 μl of a binding buffer (water with 0.1 g / ml PEG 6000) was added to the above solution. c) A 1 mL molded pipette tip packed with 10 mg cotton (cotton tip, as shown in Figure 3 [c]) was placed to interact with the cotton. d) Then, the cotton tip was specifically washed with 1 ml of wash buffer 1 (50% ethanol) and 2 ml of wash buffer 2 (50% ethanol containing 50 mM MgCy. e) The cotton tip was washed with water (3 X 1mL). f) Nucleic acids were eluted in 200 µL of elution buffer (10 mM bicin, 10 mM KCl, pH 9.8) at 95 ° C.
    Example-10: RNA extraction from serum a) 100 μl of positive Chikungunya serum was added to 150 μl of lysed buffer (40 μl of 10 mg / ml proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 2 min. b) 300 μl of a binding buffer (water with 0.1 g / ml PEG6000) was added to the above solution. c) A molded 1 mL pipette tip packed with 10 mg of cotton (cotton tip, as shown in Figure 3 [c]) was placed to interact with the cotton. d) Then, the cotton tip was washed specifically with 3 ml of wash buffer 1 (50% ethanol containing 50 mM MgCl2). e) The cotton tip was washed with water (3X1 mL). f) Nucleic acids were eluted in 200 µL of elution buffer (10 mM bicin, 10 mM KCl, pH 9.8) at 95 ° C.
    Example-11: Extraction of phlegm DNA a) 50 μl of phlegm was added to 150 μl of lysed buffer (40 μl of 10 mg / ml proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100, pH 9.5). The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 85 ° C for 6 min. b) 150 μl of a binding buffer (water with 0.1 g / ml PEG 6000) was added to the above solution. c) 10 mg of cotton were placed to interact with the above solution. d) Then, the cotton was specifically washed with 3 ml of wash buffer 1 (50% ethanol containing 50 mM MgCl2). e) The cotton was washed with water (3 X 1mL). f) Nucleic acids were eluted in 100 µL elution buffer (10 mM bicin, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-12: Extraction of phlegm DNA a) 50 μl of phlegm were added to 75 μl of lysed buffer (40 OL of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100, pH 9.5). The resulting solution was heated to 60 ° C and left at that temperature for 5 min. b) 150 μl of a binding buffer (water with 0.1 g / ml PEG 6000) was added to the above solution. c) 10 mg cotton were placed to interact with the solution above. d) Then, the cotton was washed specifically with 3 ml of wash buffer 1 (50% ethanol containing 100 mM MgCl2). e) The cotton was washed with water (3 X 1mL). f) Nucleic acids were eluted in 100 µL elution buffer (10 mM bicin, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-13: RNA extraction from tissue a) 50 μl of rabies positive tissue was added to 175 μl of lysed buffer (40 μl of 10 mg / mL proteinase K, 5.6 M guanidine thiocyanate, 100 mM EDTA, 20 mM Tris, 0.01% triton X-100, pH 9.5), vortexed for 7 min and the supernatant was transferred to a tube. The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 75 ° C for 3 min. b) 350 μl of a binding buffer (water with 0.1 g / ml PEG 6000) was added to the above solution. c) 20 mg of cotton were placed to interact with the above solution. d) Then, the cotton was washed specifically with 3 ml of wash buffer 1 (50% ethanol containing 100 mM MgCl2). e) The cotton was washed with water (3 X 1mL). f) Nucleic acids were eluted in 100 µL of elution buffer (10 mM bicin, 10 mM KCI, pH 9.8) at 95 ° C.
    Example-14: RNA extraction from blood a) Lysed buffer, binding buffer, wash buffer and elution buffer were prepared in DEPC water. b) 50 μl of Chikungunya blood were placed in 50 μl of 10 mg / mL proteinase K and 250 μl lysed buffer (5.6 M guanidine thiocyanate, 20 mM EDTA, 20 mM Tris, 100 mM MgCI2, 0.1% triton X-100). The tube was heated to 60 ° C and left at that temperature for 3 min. Then, the tube was heated to 80 ° C for 2 min. c) 1 ml of a binding buffer (10% PEG 6000) was added to the above solution. d) A 3 mL syringe packaged with cotton (cotton syringe, as shown in Figure 1 [a]) was placed to interact with the solution, pulling the syringe plunger back and forth five times. e) Then, the cotton syringe was specifically washed with 3 mL of wash buffer 1 (50% ethanol containing 100mM MgCL) by pulling the syringe plunger back and forth seven times. f) The cotton syringe was washed with water (3X2 mL) by pulling the syringe plunger back and forth with the liquid three times with each wash. g) The nucleic acids were eluted in 200 μl of elution buffer (10 mM bicine, 10 mM KCl, pH 9.8) at 95 ° C by pulling the syringe plunger back and forth with the liquid twice. h) The nucleic acids present in the blood were obtained in ready PCR form and the complete protocol took about 9 minutes.
    Example-15: Extraction of peptide nucleic acids (PNA) a) 50 μl of PNA containing standard solution was added to 75 μl of lysed buffer (10 μl of 10 mg / ml proteinase K, 5.6 M guanidine hydrochloride, 100 mM EDTA, 20 mM Tris, 0.01% X-100 triton, pH 9.5), vortexed for 7 min, the supernatant being transferred to a tube. The resulting solution was heated to 60 ° C and left at that temperature for 3 min. Then, the solution was heated to 75 ° C for 3 min. b) 150 μl of a binding buffer (water with 0.1 g / ml PEG6000) was added to the above solution. c) 10 mg of cotton were placed to interact with the above solution. d) Then, the cotton was washed specifically with 3 ml of wash buffer 1 (50% ethanol containing 100 mM MgCl2). e) The cotton was then washed with water (3 X 1mL). f) Proteinic nucleic acids were eluted in 100 µL of elution buffer (10 mM bicin, 10 mM KCl, pH 9.8) at 95 ° C.
    Example-16: PCR amplification The DNA / RNA samples purified by the protocol of the present disclosure are subjected to PCR amplification followed by gel electrophoresis. The results are shown in figures 4, 5 6, and 7. Figure 4 provides comparative bands of isolated and purified DNA samples using viscose, commercial viscose swabs, cotton packed in 1 mL pipette tip, commercial silica column and cotton swab. Similarly, figure 5 provides comparative bands of DNA samples purified by different protocols, that is, cotton packed in a 1 mL pipette tip, cotton packed in a 2 mL syringe, commercial silica column, and molecular weight marker .
    Also, figure 6 provides comparative bands of DNA samples purified by different protocols, that is, molecular weight marker, commercial silica protocol, cotton packed in 1 mL pipette tip, Whatman filter paper No. 1 packed in tip pipette and FTA card protocol.
    In addition, Figure 7 provides comparative bands of a 30 ct RNA sample amplified by RT-PCR, which have been purified by different protocols. The protocols used different sources of the cotton matrix, namely, surgical cotton, autoclaved cotton, cotton washed with sodium hydroxide, cotton washed with hydrochloric acid and absorbent cotton.
    Although several aspects and achievements have been revealed in the present, other aspects and achievements will become apparent to non-subject technicians. The various aspects and achievements disclosed herein are for illustrative purposes and should not be construed as limiting, with the true scope and spirit being indicated by the following claims.
    权利要求:
    Claims (28)
    [0001]
    1. METHOD, for the isolation of nucleic acid from a sample, said method comprising the steps of: (a) Adding a lysed buffer with basic pH of the sample containing nucleic acid to obtain a lysed solution; or (b) Adding a lysed buffer with basic pH in combination with a sample binding buffer to obtain a lysed solution (c) Adding a binding buffer to the solution obtained in step (a) to bind the nucleic acid to a cotton matrix or direct connection of the solution from step (b) to a cotton matrix; characterized by the binding pH being 8 to 11; and (d) washing with washing buffer and eluting the nucleic acid bound to the cotton matrix with elution buffer to isolate and purify the nucleic acid, wherein the lysed buffer comprises guanidine thiocyanate or guanidine hydrochloride, EDTA, Tris, a detergent and, optionally, urea, a polyol, a monovalent salt containing the IA group cation and / or divalent salt containing a protein digesting enzyme and group IIA cation.
    [0002]
    2. METHOD according to claim 1, characterized in that said nucleic acid is selected from a group consisting of DNA, RNA and PNA.
    [0003]
    METHOD, according to claim 1, characterized in that said sample is a biological or non-biological sample.
    [0004]
    4. METHOD according to claim 3, characterized in that the biological sample is selected from a group consisting of blood, phlegm, serum, saliva or tissue extracts with the non-biological sample being selected from a group comprising chemically synthesized PNA.
    [0005]
    5. METHOD, according to claim 1, characterized in that the lysis is carried out at a pH between 8 to 11.
    [0006]
    6. METHOD according to claim 1, characterized in that said EDTA has a concentration ranging from 10 mM to 300 mM.
    [0007]
    METHOD according to claim 1, characterized in that said guanidine thiocyanate or said guanidine hydrochloride has a concentration ranging from 0.1 M to 7 M.
    [0008]
    8. METHOD, according to claim 1, characterized in that said urea has a concentration ranging between 0.01 M to 7 M.
    [0009]
    9. METHOD according to claim 1, characterized in that said Tris has a concentration ranging from 0.01 mM to 100 mM.
    [0010]
    10. METHOD according to claim 1, characterized in that said polyol has a concentration ranging from 0.01% to 30% (v / v).
    [0011]
    11. METHOD according to claim 1, characterized in that said detergent is selected from a group consisting of sodium lauryl sulfate, sodium dodecyl sulfate, Triton X-100, NP-40 and Tween 20 or any of their combinations and that the protein digesting enzyme is proteinase K.
    [0012]
    METHOD, according to claim 1, characterized in that said binding buffer is water, optionally together with polyols or non-polyols.
    [0013]
    METHOD according to claim 12, characterized in that said polyol comprises water-soluble polyol compounds selected from a group consisting of polyethylene glycol, glycerol, polypropylene glycol, ethylene glycol and propylene glycol.
    [0014]
    METHOD according to claim 12, characterized in that said non-polyol comprises alcohols selected from a group consisting of methanol, ethanol, propanol or any water-soluble liquid with a functional group of acid, amine, alcohol, phenol , amide or ester as one of the functional groups; or any of its combinations.
    [0015]
    METHOD according to claim 1, characterized in that said wash comprises a first wash with a wash buffer comprising 1% to 99% (v / v) aqueous alcohol followed by multiple washes with a wash buffer comprising 100% of water.
    [0016]
    16. METHOD according to claim 15, characterized in that said aqueous alcohol is selected from a group comprising ethanol, methanol, n-propanol, 2-propanol, glycerol, PEG, PPG, ethylene glycol and propylene glycol.
    [0017]
    17. METHOD, according to claim 1 or 15, characterized in that said water is selected from a group consisting of deionized water, DNase-free water, RNase-free water, MilliQ water, filtered water, tap water and soil water or any of its combinations.
    [0018]
    18. METHOD according to claim 1 or 15, characterized in that said washing buffer can ideally comprise salts selected from a group consisting of MgCl2, CaCl2, NaCl and KCl, or buffers selected from a group consisting of bicin, tricine, Tris , HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bis-tris, PIPES, ACES, BES, TES, borate, TAPS, CHES, CAPS, ethanolamine and piperidine, with pH ranging between 5 to 12.
    [0019]
    19. METHOD, according to claim 1, characterized in that said elution buffer comprises hot water with temperatures varying between 45oC to 99oC together with buffer or salt, having pH ranging between 8 to 11.
    [0020]
    20. METHOD, according to claim 19, characterized in that said water is selected from a group consisting of deionized water, DNase-free water, RNase-free water, MilliQ water, filtered water, tap water and soil water or any of your combinations.
    [0021]
    21. METHOD according to claim 19, characterized in that said buffer is selected from a group consisting of bicin, tricine, Tris, HEPES, CHAPS, phosphate, acetate, MES, pyridine, piperazine, Bis-tris, PIPES, ACES, BES, TES, borate, TAPS, CHES, CAPS, ethanolamine and piperidine, with pH ranging from 5 to 12.
    [0022]
    22. METHOD according to claim 19, characterized in that said salt is selected from a group consisting of MgCl2, CaCl2, NaCl and KCl or any of their combinations in the concentration ranging from 0.01 mM to 100 mM.
    [0023]
    23. METHOD according to claim 1, characterized in that the cotton is selected from a group consisting of natural cotton, surgical cotton, clinical cotton, commercial cotton, woven cotton, cotton washed with water, cotton washed with acid or base, autoclaved cotton, cotton treated with buffer with pH ranging from 1 to 14, cotton treated with saline solution, cotton treated with organic solvent, pressed cotton and processed cotton.
    [0024]
    24. KIT, for isolating nucleic acid from a sample, said kit characterized by comprising a cotton matrix, lysed buffer with basic pH, binding buffer in which the binding pH is 8 to 11, washing buffer and buffer elution method, in which the lysed buffer comprises guanidine thiocyanate or guanidine hydrochloride, EDTA, Tris, a detergent and optionally urea, a polyol, a monovalent salt containing the group IA cation and / or divalent salt containing a group IIA cation and protein digesting enzyme.
    [0025]
    25. KIT according to claim 24, characterized in that the cotton is selected from a group consisting of natural cotton, surgical cotton, clinical cotton, commercial cotton, woven cotton, cotton washed with water, cotton washed with acid or base, autoclaved cotton , cotton treated with buffer with pH ranging from 1 to 14, cotton treated with saline solution, cotton treated with organic solvent, pressed cotton and processed cotton.
    [0026]
    26. KIT according to claim 24, characterized in that said buffer is selected from a group consisting of lysed buffer according to claims 5 to 11, binding buffer according to claims 12 to 14, wash buffer according to claims 15 and 19, and elution buffer according to claims 15 and 20 to 23.
    [0027]
    27. KIT according to claim 24, characterized in that said sample comprises biological or non-biological samples.
    [0028]
    28. KIT according to claim 27, characterized in that the biological sample is selected from a group consisting of blood, phlegm, serum, saliva or tissue extracts and the non-biological sample is selected from the group consisting of chemically synthesized PNA.
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    ZA201205053B|2013-03-27|
    CY1119878T1|2018-06-27|
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    JP5791631B2|2015-10-07|
    DK2521780T3|2017-12-04|
    PT2521780T|2017-12-21|
    LT2521780T|2018-02-12|
    JP2013516186A|2013-05-13|
    CN102725407B|2016-09-14|
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    法律状态:
    2017-09-05| B12F| Appeal: other appeals|
    2020-04-07| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|
    2020-09-24| B09A| Decision: intention to grant|
    2020-12-01| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/01/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    IN50CH2010|2010-01-07|
    IN50/CHE/2010|2010-01-07|
    PCT/IB2011/050044|WO2011083429A1|2010-01-07|2011-01-06|A method for isolation of nucleic acids and a kit thereof|
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