CN111733156B - Low-concentration DNA protective agent - Google Patents
Low-concentration DNA protective agent Download PDFInfo
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- CN111733156B CN111733156B CN202010587350.8A CN202010587350A CN111733156B CN 111733156 B CN111733156 B CN 111733156B CN 202010587350 A CN202010587350 A CN 202010587350A CN 111733156 B CN111733156 B CN 111733156B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- 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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Abstract
The invention discloses a low-concentration DNA protective agent, and the low-concentration DNThe A protective agent consists of pH buffer solution, nuclease inhibitor and anti-adsorption agent, and can effectively prevent the concentration of the A protective agent from being as low as 2 x 10‑4And the degradation of the DNA of pM prevents the adsorption of the DNA storage tube to the low-concentration DNA, thereby ensuring that the low-concentration DNA meets the quantitative requirement of the experiment, ensuring the experiment operation to be more accurate, reducing the unnecessary consumption of the DNA caused by adsorption and degradation, and correspondingly reducing the experiment cost.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a low-concentration DNA protective agent.
Background
DNA is a stable macromolecule having a double helix structure, and DNA at a high concentration can be stably stored in a refrigerator at-20 ℃ or-80 ℃, but DNA at a low concentration is easily degraded or otherwise lost, and accurate quantification of DNA is also affected due to adsorption of DNA by a storage container when the DNA concentration is low.
Accordingly, further developments and improvements are still needed in the art.
Disclosure of Invention
In order to solve the above-mentioned problems, a low-concentration DNA protecting agent has been proposed which is effective not only in preventing degradation of DNA at a low concentration but also in preventing adsorption of DNA to a DNA storage tube.
In order to achieve the purpose, the invention provides the following technical scheme:
a low concentration DNA protectant comprises pH buffer solution, nuclease inhibitor and anti-adsorption agent.
Preferably, the pH buffer is a tris buffer.
Preferably, the tris buffer has a concentration of 10mM to 100mM and a pH of 7.5 to 9.0.
Preferably, the concentration of the nuclease inhibitor is 0.1-10%.
Preferably, the nuclease inhibitor is one or more of alcohol, DTT and protease K, EDTA.
Preferably, the concentration of the anti-adsorption agent is 0.01% -3%.
Preferably, the anti-adsorption agent is one or more of polyvinyl alcohol, trehalose, betaine, Tween20 and glycerol.
Further, the nuclease inhibitor consists of polyvinyl alcohol and Tween 20.
Preferably, D isNA concentration of 2X 10 or more-4pM。
Has the advantages that:
the present invention provides a low concentration DNA protectant which is effective against DNA damage at concentrations as low as 2X 10-4And the degradation of the DNA of pM prevents the adsorption of the DNA storage tube to the low-concentration DNA, thereby ensuring that the low-concentration DNA meets the quantitative requirement of the experiment, ensuring the experiment operation to be more accurate, reducing the unnecessary consumption of the DNA caused by adsorption and degradation, and correspondingly reducing the experiment cost.
Detailed Description
In order to make the technical solutions of the present invention better understood, the following description is provided clearly and completely, and other similar embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present application based on the embodiments in the present application.
The following are specific embodiments of the present invention:
preparing pH buffer solution, nuclease inhibitor and anti-adsorption agent into 11 different low-concentration DNA protective agents, which respectively comprise the following components:
protective agent 1: 10mM tris, 5% glycerol, 0.05% Tween20, 5% proteinase K;
protective agent 2: 10mM tris, 0.05% Tween-20, 1% ethanol;
protective agent 3: 10mM tris, 0.465% DTT, 1% betaine;
protective agent 4: 10mM tris, 0.5% polyvinyl alcohol, 0.5% DTT;
and (5) a protective agent: 20mM phosphate buffer, 3, 0.075% Tween20, 0.03% glycerol, 0.1% ethanol;
protective agent 6: 10mM tris, 0.075% Tween20, 0.1% betaine, 9.3% DTT;
protective agent 7: 20mM carbonate buffer, 0.075% Tween20, 0.03% glycerol, 3% proteinase K;
protective agent 8: 10mM tris, 0.2% Tween20, 0.1% glycerol, 2% ethanol;
protecting agent 9: 10mM tris, 0.075% Tween20, 0.5 EDTA;
protective agent 10: 10mM tris, 0.075% Tween20, 0.292% EDTA;
protective agent 11: 10mM tris, 0.075% Tween20, 0.1% betaine, 5% proteinase K.
Meanwhile, selecting a DNA fragment with a known sequence, designing a primer, and carrying out PCR to obtain a PCR product for quantification: DNA was diluted to 1000pM with 500ml of sterile water in life 10977015-;
the DNAs were diluted with a gradient of 83pM, 8.3X 10, respectively, using 1-11 protectants, respectively-1pM、8.3×10-2pM、8.3×10-3pM、8.3×10-4pM, will dilute DNA store in nuclease-free low adsorption 1.5ml centrifuge tube;
the DNA gradient concentration is measured by using an ABI7500qPCR instrument to obtain the CT difference value of each gradient concentration of the DNA, wherein the CT difference value is 3.0-3.6 to indicate that the DNA has no obvious change, the DNA concentration is changed when the CT difference value exceeds the range, and the larger the deviation is, the larger the concentration change is.
The following are specific examples of different storage conditions and different storage times:
example 1:
the DNA concentrations stored in the protecting agents 1 to 11 were measured after storing at 4 ℃ for 1 day, and the results are shown in Table 1:
TABLE 1
As is clear from Table 1, the protecting agents 8, 9 and 10 were able to effectively protect DNA at each concentration gradient when stored at 4 ℃ for 1 day.
Based on the results of example 1, protective agents 8, 9 and 10 with better protective effects were selected for subsequent experiments.
Example 2:
after freezing and thawing the DNA for 30 times, testing the protection effect of different protective agents on the DNA under extreme conditions, and the test results are shown in Table 2:
TABLE 2
As can be seen from Table 2, the protective agent 8 can effectively protect DNA of each concentration gradient under the extreme conditions of repeated freeze thawing for 30 times.
Example 3:
after the DNA was stored at 4 ℃ for 20 days, the DNA was measured for each concentration gradient, and the measurement results are shown in Table 3:
TABLE 3
As is clear from Table 3, the protective agent 8 was effective in protecting DNA at each concentration gradient when stored at 4 ℃ for 20 days.
Example 4:
after the DNA was stored at room temperature (25 ℃) for 7 days, the DNA was measured for each concentration gradient, and the results of the measurements are shown in Table 4:
TABLE 4
As can be seen from Table 4, the protective agent 8 can effectively protect DNA at each concentration gradient after being stored at room temperature for 7 days.
Example 5:
the DNA was stored at-20 ℃ for one year, and the DNA was measured for each concentration gradient, and the results are shown in Table 5:
TABLE 5
As can be seen from Table 5, the protective agent 8 can effectively protect DNA of each concentration gradient when stored at-20 ℃ for one year.
The present invention has been described in detail, and it should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Claims (1)
1. A low-concentration DNA protective agent is characterized by comprising a pH buffer solution, a nuclease inhibitor and an anti-adsorption agent; the pH buffer solution is tris buffer solution with the concentration of 10 mM; the nuclease inhibitor is alcohol, and the concentration is 2%; the anti-adsorption agent comprises Tween20 and glycerol, wherein the concentration of Tween20 is 0.2%, and the concentration of glycerol is 0.1%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010587350.8A CN111733156B (en) | 2020-06-24 | 2020-06-24 | Low-concentration DNA protective agent |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010587350.8A CN111733156B (en) | 2020-06-24 | 2020-06-24 | Low-concentration DNA protective agent |
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| Publication Number | Publication Date |
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| CN111733156A CN111733156A (en) | 2020-10-02 |
| CN111733156B true CN111733156B (en) | 2021-12-14 |
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| CN117737048A (en) * | 2022-12-29 | 2024-03-22 | 南京诺唯赞动物保健有限公司 | DNA preservation solution |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559654A (en) * | 2010-12-17 | 2012-07-11 | 上海元基融生物科技有限公司 | DNA (deoxyribonucleic acid) preserving solvent and DNA preserving method |
| CN106065400A (en) * | 2015-04-24 | 2016-11-02 | 上海润腾生物科技有限公司 | A kind of ribonucleic acid protective agent, test kit, application and store method |
| CN108893524A (en) * | 2018-06-04 | 2018-11-27 | 北京启衡星生物科技有限公司 | The protective agent of dissociative DNA in blood plasma |
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- 2020-06-24 CN CN202010587350.8A patent/CN111733156B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559654A (en) * | 2010-12-17 | 2012-07-11 | 上海元基融生物科技有限公司 | DNA (deoxyribonucleic acid) preserving solvent and DNA preserving method |
| CN106065400A (en) * | 2015-04-24 | 2016-11-02 | 上海润腾生物科技有限公司 | A kind of ribonucleic acid protective agent, test kit, application and store method |
| CN108893524A (en) * | 2018-06-04 | 2018-11-27 | 北京启衡星生物科技有限公司 | The protective agent of dissociative DNA in blood plasma |
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| CN111733156A (en) | 2020-10-02 |
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