CN112226490A - Lysis buffer solution for DNA extraction - Google Patents
Lysis buffer solution for DNA extraction Download PDFInfo
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- CN112226490A CN112226490A CN202011261621.7A CN202011261621A CN112226490A CN 112226490 A CN112226490 A CN 112226490A CN 202011261621 A CN202011261621 A CN 202011261621A CN 112226490 A CN112226490 A CN 112226490A
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- dna
- lysis buffer
- buffer solution
- ches
- dna extraction
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- 239000012139 lysis buffer Substances 0.000 title claims abstract description 32
- 238000007400 DNA extraction Methods 0.000 title claims abstract description 14
- 239000000243 solution Substances 0.000 title abstract description 9
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical group [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 8
- MKWKNSIESPFAQN-UHFFFAOYSA-N N-cyclohexyl-2-aminoethanesulfonic acid Chemical compound OS(=O)(=O)CCNC1CCCCC1 MKWKNSIESPFAQN-UHFFFAOYSA-N 0.000 claims description 6
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 239000008062 guanidine hydrochloride buffer Substances 0.000 claims description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical class NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 claims 2
- 239000000872 buffer Substances 0.000 claims 2
- 239000003599 detergent Substances 0.000 claims 2
- 238000001514 detection method Methods 0.000 abstract description 14
- 239000003112 inhibitor Substances 0.000 abstract description 10
- 239000011324 bead Substances 0.000 abstract description 7
- 229910052710 silicon Inorganic materials 0.000 abstract description 7
- 239000010703 silicon Substances 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 102000001554 Hemoglobins Human genes 0.000 abstract description 4
- 108010054147 Hemoglobins Proteins 0.000 abstract description 4
- QJYQSFRDJBQMCT-UHFFFAOYSA-N cyclohexanamine;ethanesulfonic acid Chemical compound CCS(O)(=O)=O.NC1CCCCC1 QJYQSFRDJBQMCT-UHFFFAOYSA-N 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract 2
- 150000002357 guanidines Chemical class 0.000 abstract 1
- 238000000605 extraction Methods 0.000 description 7
- 229960000789 guanidine hydrochloride Drugs 0.000 description 7
- 239000000523 sample Substances 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101100281953 Homo sapiens GAPDH gene Proteins 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a lysis buffer solution for DNA extraction, which comprises a guanidine salt buffer solution and 2-cyclohexylamine ethanesulfonic acid. When the buffer solution is used for DNA extraction, protein PCR inhibitors in a sample, such as hemoglobin, mucin and the like, cannot be adsorbed on the surface of the silicon hydroxyl magnetic bead together with DNA, the content of the PCR inhibitors in the extracted DNA is low, the repeatability of a PCR detection result can be effectively improved, and the false negative result of detection can be reduced.
Description
Technical Field
The invention relates to a lysis buffer solution for DNA extraction and an extraction kit.
Background
Molecular diagnosis is a technology for diagnosing by detecting the structure or expression level of genetic material in a patient body by applying a molecular biological method, common molecular diagnosis methods comprise PCR, isothermal amplification, sequencing and the like, wherein PCR is the molecular diagnosis method with the widest application range at present. Compared with other diagnostic methods, molecular diagnosis has the advantages of high detection sensitivity, high accuracy and strong specificity. The detection target of the molecular diagnostic reagent is nucleic acid (DNA or RNA), and before detection, nucleic acid extraction needs to be performed on a sample to be detected.
In the extraction of DNA from a sample (e.g., blood, sputum, swab), a lysis buffer is generally prepared using guanidine hydrochloride as a main raw material. Guanidine hydrochloride can crack cells and release DNA, and the DNA is extremely stable in a high-concentration guanidine hydrochloride environment and can be adsorbed on the surface of silicon hydroxyl magnetic beads. After washing off guanidine hydrochloride by using a special washing buffer solution, dissolving DNA from the surface of the silicon hydroxyl magnetic beads by using an elution buffer solution, and finishing the extraction of the DNA.
In the high-concentration guanidine hydrochloride solution, not only DNA is easily adsorbed on the surface of the silicon hydroxyl magnetic bead, but also common protein PCR inhibitors in a sample, such as hemoglobin, mucin and the like are easily adsorbed on the surface of the silicon hydroxyl magnetic bead. And these PCR inhibitors are not easily removed by the washing buffer and are eventually eluted together with the DNA by the elution buffer. When the DNA containing the PCR inhibitor is used for PCR detection, false negative test results are often obtained because the polymerase activity is inhibited.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a lysis buffer and an extraction kit for DNA extraction.
The invention solves the technical problems through the following technical scheme:
a lysis buffer for DNA extraction is characterized in that 2-cyclohexylaminoethanesulfonic acid (CHES) is added on the basis of a conventional guanidine hydrochloride buffer.
In the scheme, when the lysis buffer is used for DNA extraction, common protein PCR inhibitors in a sample, such as hemoglobin and mucin, cannot be adsorbed on the surface of the silicon hydroxyl magnetic bead together with DNA, the content of the PCR inhibitors in the extracted DNA is low, and the false negative result of PCR detection can be effectively reduced.
Preferably, the concentration of 2-cyclohexylaminoethanesulfonic acid (CHES) in the lysis buffer is 1 mM-100 mM.
Preferably, the lysis buffer further comprises other conventional components, such as guanidine hydrochloride with a concentration of 1M-8M, Triton X-100 with a concentration of 0.1-5%, and the like.
A DNA extraction kit comprises the lysis buffer.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The positive progress effects of the invention are as follows: the method comprises the steps of adding 1-100 mM of 2-cyclohexylamine ethanesulfonic acid (CHES) into a lysis buffer solution for DNA extraction; so that common protein PCR inhibitors in a sample, such as hemoglobin, mucin and the like, cannot be adsorbed on the surface of the silicon hydroxyl magnetic bead together with DNA, the content of the PCR inhibitors in the extracted DNA is low, the repeatability of a PCR detection result can be effectively improved, and the false negative result of the detection can be reduced
Drawings
FIG. 1 shows the results of DNA detection from the lysis buffer (CHES +) and the conventional lysis buffer (CHES-) in example 1.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: preparing lysis buffer
1) Preparation of CHES-free lysis buffer (CHES-)
750mL of 8M guanidine hydrochloride, 50mL of 1M Tris-HCl (pH 7.5), 50mL of 500mM EDTA, 100mL of 5M NaCl and 50mL of Triton X-100 were weighed and mixed well to obtain a lysis buffer (CHES-) containing no CHES.
1) Preparation of lysis buffer containing CHES (CHES +)
To the aforementioned CHES-free lysis buffer (CHES-) was added 2-cyclohexylaminoethanesulfonic acid (CHES) to give a final concentration of 7.5mM, thereby giving a CHES-containing lysis buffer (CHES +).
Example 2: comparison of the DNA extraction efficiency of the two lysis buffers
10 blood samples were lysed with two lysates (CHES-) and (CHES +) respectively to extract DNA. The procedure for extracting DNA was identical, the same wash buffer used in the extraction was 75% ethanol, and the same elution buffer used in the extraction was 10mM Tris (pH 8.0).
The extracted DNA was detected by real-time fluorescent PCR using human GAPDH gene detection primers/probes.
The results are shown in the following table:
the experimental result shows that the amplification curve of the DNA extracted by the CHES-containing lysis buffer (CHES +) is closer to the front (the Ct value is smaller) and the amplification curve is more concentrated (the Ct value is similar), which indicates that the DNA extracted by the CHES-containing lysis buffer has less interfering substances and good experimental repeatability. The DNA extracted by the lysis buffer (CHES-) without CHES has a more backward amplification curve (larger Ct value) and a more dispersed amplification curve (larger difference of Ct value), even has the condition of no amplification signal (NoCt), which shows that the DNA extracted by the lysis buffer has more interfering substances, the experimental result is unstable, and even false negative result can occur due to interference.
The detection result shows that when the lysis buffer solution is used for DNA extraction, the content of PCR inhibitors in the extracted DNA is low, the repeatability of the PCR detection result can be effectively improved, and the false negative result of the detection can be reduced.
Claims (5)
1. A lysis buffer for DNA extraction is characterized in that the lysis buffer comprises guanidine hydrochloride buffer and 2-cyclohexylaminoethanesulfonic acid.
2. The lysis buffer of claim 1, wherein the concentration of 2-cyclohexylaminoethanesulfonic acid (CHES) in the buffer is between 1mM and 100 mM.
3. The lysis buffer of claim 2, further comprising a guanidinium salt at a concentration of 1M to 8M and/or 0.1 to 10% detergent.
4. The lysis buffer of claim 3 wherein the guanidinium salt in the buffer is guanidinium hydrochloride and the detergent is Triton X-100.
5. A kit for DNA extraction, comprising the lysis buffer of any one of claims 1 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011261621.7A CN112226490A (en) | 2020-11-12 | 2020-11-12 | Lysis buffer solution for DNA extraction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011261621.7A CN112226490A (en) | 2020-11-12 | 2020-11-12 | Lysis buffer solution for DNA extraction |
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| Publication Number | Publication Date |
|---|---|
| CN112226490A true CN112226490A (en) | 2021-01-15 |
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Family Applications (1)
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|---|---|---|---|
| CN202011261621.7A Pending CN112226490A (en) | 2020-11-12 | 2020-11-12 | Lysis buffer solution for DNA extraction |
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| Country | Link |
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| CN (1) | CN112226490A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996000228A1 (en) * | 1994-06-23 | 1996-01-04 | Dade International Inc. | Method for the rapid isolation of nucleic acid |
| CN102046793A (en) * | 2008-05-30 | 2011-05-04 | 恰根有限公司 | Lysis, binding and/or wash reagent for isolating and/or purifying nucleic acids |
| CN103890176A (en) * | 2011-09-06 | 2014-06-25 | 西北大学 | A method of preparing biological material |
| EP3135769A1 (en) * | 2015-08-26 | 2017-03-01 | Qiagen GmbH | Kits and methods for extracting rna |
| CN108624586A (en) * | 2018-03-22 | 2018-10-09 | 重庆中元汇吉生物技术有限公司 | A kind of nucleic acid extraction kit and its application process |
-
2020
- 2020-11-12 CN CN202011261621.7A patent/CN112226490A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996000228A1 (en) * | 1994-06-23 | 1996-01-04 | Dade International Inc. | Method for the rapid isolation of nucleic acid |
| CN102046793A (en) * | 2008-05-30 | 2011-05-04 | 恰根有限公司 | Lysis, binding and/or wash reagent for isolating and/or purifying nucleic acids |
| CN103890176A (en) * | 2011-09-06 | 2014-06-25 | 西北大学 | A method of preparing biological material |
| EP3135769A1 (en) * | 2015-08-26 | 2017-03-01 | Qiagen GmbH | Kits and methods for extracting rna |
| CN108624586A (en) * | 2018-03-22 | 2018-10-09 | 重庆中元汇吉生物技术有限公司 | A kind of nucleic acid extraction kit and its application process |
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