CN102559654A - DNA (deoxyribonucleic acid) preserving solvent and DNA preserving method - Google Patents
DNA (deoxyribonucleic acid) preserving solvent and DNA preserving method Download PDFInfo
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- CN102559654A CN102559654A CN2010105950553A CN201010595055A CN102559654A CN 102559654 A CN102559654 A CN 102559654A CN 2010105950553 A CN2010105950553 A CN 2010105950553A CN 201010595055 A CN201010595055 A CN 201010595055A CN 102559654 A CN102559654 A CN 102559654A
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Abstract
The invention discloses a DNA (deoxyribonucleic acid) preserving solvent and a DNA preserving method. The DNA preserving solvent comprises glycerol, TE buffer solution and 1-carboxyl-N, N, N-trimethyl-B lactone, wherein the final volume of the glycerol is 25-80%, the final concentration of the TE buffer solution is 1*TE-5*TE, and the final concentration of the 1-carboxyl-N, N, N-trimethyl-B lactone is 1-6mol/L. The DNA preserving method comprises the following steps of: (1) extracting DNA; and (2) dissolving the DNA in the DNA preserving solvent to be preserved. The DNA preserving solvent disclosed by the invention has the advantages of simple preparation, low cost, good preserving effect and long shelf life and is prevented from freezing and thawing repeatedly. The invention provides initial DNA records for genomics research, identity authentication, gene diagnosis, gene therapy, organ transplantation and the like in the future.
Description
Technical field
The present invention relates to the preservation of a kind of DNA (deoxyribonucleic acid, thymus nucleic acid), particularly relate to a kind of DNA and preserve solvent and store method thereof.
Background technology
DNA is that chromosomal important composition is abundant, is Eukaryotic hereditary base substance, contains the individual genetic information of species, can be used for making up genomic library, separates required gene and detects relevant molecule marker etc.The genetic information amount contained owing to DNA is big, and obtains easily, has extremely stable chemical property, is one of main contents of collection of present germ plasm resource and genetic resources and preservation.
The sequential structure of dna molecular has three key properties: (1) Different Individual sequence is different; (2) constant throughout one's life; (3) dna sequence dna in the cell of the variant position of same human body is identical.Therefore dna molecular itself possesses the characteristic of archives, promptly has permanent stability, information attribute, individual specificity and attribute that can be for future reference.
In fact, in research fields such as disease gene group, pharmacogenomics, population genetics and evolution, DNA is the basic material of the most reliable evidence and research process, is widely used.Simultaneously the life science person recognizes day by day and preserves DNA for the importance of the development of system biological medical science from now on, and various countries begin to preserve DNA one after another.What early start implemented that extensive DNA preserves in the world is the Decode company of Iceland, and all DNA sample extremely related data nearly comes from Iceland grownup of 1/3 and 90% above Iceland the elderly.Utilize the biological specimen storehouse of its foundation, Decode has found the Disease-causing gene of more than 20 kind of diseases such as myocardial infarction.National research units such as Britain, the U.S., Israel have set up similar genetic resources sample storehouse subsequently, and people's dna sample of Israel's these all informations for sale.
In order to keep the integrity of dna structure; Avoid DNA fracture and degraded on a large scale, the best store method of DNA is dry powder-80 ℃ perhaps liquid nitrogen cryogenics preservation, then needs a large amount of DNA but will process dry powder; The difficulty of drawing materials does not have operability in actual dna sample resource acquisition.And preserve DNA with absolute ethyl alcohol, though packing is in advance preserved, also there is the multigelation problem.And the solid phase DNA technology of preserving is the new ideas that propose recently, is applicable to permanent preservation, and whether the preservation matrix of this method influence the DNA subsequent experimental, preserve concrete effect, does not have any data of this respect at present, and the time that still remains tests.And for needing nonexpondable sample between preservation period, need extract DNA again, complicated operation, and cause dna break easily, will cause some can't carry out to the high test experience of DNA specification of quality.
Summary of the invention
The technical problem that the present invention will solve provides a kind of DNA and preserves solvent and store method thereof.Utilize this DNA to preserve solvent; Can be very simply, prolonged preservation DNA; And the multigelation that DNA extraction is convenient, avoid DNA, with low cost is for genomics research from now on, identity discriminating, gene diagnosis, gene therapy, organ transplantation etc. provide primary DNA record.
For solving the problems of the technologies described above, DNA of the present invention preserves solvent, and its component comprises: and glycerine, TE damping fluid (Tris-EDTA, EDTA: YD 30), the 1-carboxy-N, and N, N-trimethylammonium second lactone, wherein, the final volume of glycerine is 25%～80%; The final concentration of TE damping fluid is 1 * TE～5 * TE, and the pH value is 6～8, preferred 1 * TE, pH value 7.6; The 1-carboxy-N, N, the final concentration of N-trimethylammonium second lactone is 1～6mol/L, preferred 3mol/L.
Above-mentioned glycerine and 1-carboxy-N, N, the effect of N-trimethylammonium second lactone mainly is to keep liquid environment.
In addition, DNA store method of the present invention comprises step:
(1) extracts DNA;
Wherein, this DNA can be from any cell of biology, extract and purifying according to the process for extracting of routine;
(2) DNA being dissolved in aforesaid DNA preserves in the solvent and preserves.
Wherein, the ultimate density that DNA preserves can be 1～10 μ g/L, as about 6 μ g/L, and preferred 2～3 μ g/L.Very low like DNA preservation concentration, like direct big consumption experiment, the glycerine of preserving the solvent middle and high concentration might influence follow-up experiment, and the DNA of height preservation concentration needs further could test after the dilution in practical application, does not influence subsequent experimental.
Said storage temperature can be-20 ℃～-196 ℃.
Preserve compared with techniques with traditional DNA, the present invention has following advantage:
(1) making is simple, with low cost, only needs cryogenic freezing to preserve and gets final product;
(2) owing to contain the glycerine of high density, make DNA be in the liquid environment always, avoid the infringement of cut mechanically power, thereby dna degradation can be prevented effectively that therefore, the DNA preservation effect is good, long preservative period.
(3) because DNA is stored in the liquid environment, after from refrigerator or liquid nitrogen, taking out, need not dissolve, but just direct sampling or packing, thus avoided multigelation.
Therefore, the present invention's ability prolonged preservation DNA is as more than 20 years.
Embodiment
Glycerine in following examples, the component among the TE (Tris alkali, HCl, EDTA), 1-carboxy-N, N, N-trimethylammonium second lactone all is the commercially available prod.
Description of drawings
Accompanying drawing is the genome dna electrophoresis figure of embodiment 3, and wherein, hole 1 is a dna molecular marker, and hole 2 is the DNA that preserves according to embodiment 1 among the embodiment 3, and hole 3 is the DNA that preserves according to embodiment 2 among the embodiment 3.
One, the extraction of sample DNA
Adopt FlexiGene DNA Kit (QIAGEN, Cat.No.51206) genomic dna in the test kit extracting peripheral blood from patients with lung cancer.Concrete steps are following:
In 300 μ l blood samples, add 750 μ l Buffer FGl, turning upside down makes its mixing 5 times.Follow 12 centrifugal 1min under the 000rpm rotating speed.Outwell supernatant liquid after centrifugal, add 150 μ l Buffer FG2 and 1.5 μ l protein enzyme solutions again, vibration immediately is until deposition dissolving fully.Next centrifugal 3～5s, 65 ℃ of water-bath 5min then.After solution becomes olive-green from redness, add 150 μ l, 100% Virahol, fully put upside down centrifuge tube up and down, make its mixing, separate out until DNA, be macroscopic wire or bulk.Then 12, centrifugal 3min under the 000rpm rotating speed.Outwell supernatant liquid after centrifugal, add 150 μ l, 70% ethanol again, and vibration 5s.Then again 12, centrifugal 3min under the 000rpm rotating speed outwells supernatant liquid after centrifugal, and the natural air drying deposition all evaporates until all liquid.
Two, preserve
After in the centrifuge tube of the above-mentioned DNA of containing, adding the DNA stored frozen solvent (promptly preserving solvent temperature is 0 ℃) of 500 μ l, 37 ℃ are spent the night, and guarantee that DNA fully dissolves, and preserve in-80 ℃ of refrigerators then.
Wherein, the consisting of of DNA stored frozen solvent: final volume is that 35% glycerine, final concentration are that 1 * TE (pH7.6), final concentration are the 1-carboxy-N of 3mol/L, N, N-trimethylammonium second lactone.
Adopt the DNA method for extracting among the embodiment 1; The peripheral blood from patients with lung cancer 300 μ l that extract embodiment 1 carry out the DNA extracting, in the centrifuge tube that contains this DNA, add 1 * TE (pH7.6) of 500 μ l then, and 37 ℃ are spent the night; After guaranteeing fully dissolving, preserve in-80 ℃ of refrigerators.
DNA among embodiment 1 and the embodiment 2 preserved after 1 month; 10 and 16 embodiment 1 that name a person for a particular job of every workday take out with the DNA that embodiment 2 preserves; Placed 37 ℃ of incubators 15 minutes, embodiment 1 and DNA among the embodiment 2 are fully dissolved after, degree is preserved then-80; Continuously multigelation June, carry out accelerated tests.
The multiple freeze thawing of each negate was got the DNA 6 μ l that preserve according to embodiment 1 and embodiment 2 after 3 months, carried out electrophoresis with 0.8% (0.8g/100ml) agarose and identified that electrophoresis result is shown in Figure of description.Find out that from electrophorogram the DNA that embodiment 1 preserves is through behind continuous 3 months multigelations, hole 2 electrophoresis result show that genomic dna do not degrade; And the DNA that embodiment 2 preserves is through behind continuous 3 months multigelations, and hole 3 electrophoresis result show the disperse of genomic dna a slice, and degraded obviously.
Embodiment 4
Adopt the DNA method for extracting among the embodiment 1, with adding DNA stored frozen solvent among the mouse liver DNA that obtains, the ultimate density that makes DNA is 2 μ g/L, and 37 ℃ are spent the night, and guarantee that DNA fully dissolves, and preserve in-20 ℃ of refrigerators then.
Wherein, the consisting of of DNA stored frozen solvent: final volume is that 25% glycerine, final concentration are that 1 * TE (pH6), final concentration are the 1-carboxy-N of 6mol/L, N, N-trimethylammonium second lactone.
The dna molecular structure of being preserved in the present embodiment does not have destruction, can satisfy downstream experiment (like PCR, gene chip etc.) needs.
Embodiment 5
Adopt the DNA method for extracting among the embodiment 1, with adding DNA stored frozen solvent in the petridish cell DNA that obtains, the ultimate density that makes DNA is 3 μ g/L, and 37 ℃ are spent the night, and guarantee that DNA fully dissolves, then in-196 ℃ of preservations.
Wherein, the consisting of of DNA stored frozen solvent: final volume is that 80% glycerine, final concentration are that 5 * TE (pH8), final concentration are the 1-carboxy-N of 1mol/L, N, N-trimethylammonium second lactone.
The dna molecular structure of being preserved in the present embodiment does not have destruction, can satisfy downstream experiment (like PCR, gene chip etc.) needs.
Embodiment 6
Adopt the DNA method for extracting among the embodiment 1, with adding DNA stored frozen solvent in the people's liver dna that obtains, the ultimate density that makes DNA is 10 μ g/L, and 37 ℃ are spent the night, and guarantee that DNA fully dissolves, then in-120 ℃ of preservations.
Wherein, the consisting of of DNA stored frozen solvent: final volume is that 60% glycerine, final concentration are that 2 * TE (pH7.5), final concentration are the 1-carboxy-N of 5mol/L, N, N-trimethylammonium second lactone.
The dna molecular structure of being preserved in the present embodiment does not have destruction, can satisfy downstream experiment (like PCR, gene chip etc.) needs fully.
Claims (8)
1. a DNA preserves solvent, it is characterized in that: the component that this DNA preserves solvent comprises: glycerine, TE damping fluid, 1-carboxy-N, N; N-trimethylammonium second lactone, wherein, the final volume of glycerine is 25%～80%; The final concentration of TE damping fluid is 1 * TE～5 * TE; The 1-carboxy-N, N, the final concentration of N-trimethylammonium second lactone is 1～6mol/L.
2. DNA as claimed in claim 1 preserves solvent, and it is characterized in that: the final concentration of said TE damping fluid is 1 * TE, and the pH value is 6～8.
3. DNA as claimed in claim 2 preserves solvent, and it is characterized in that: the pH value of said TE damping fluid is 7.6.
4. DNA as claimed in claim 1 preserves solvent, it is characterized in that: said 1-carboxy-N, and N, the final concentration of N-trimethylammonium second lactone is 3mol/L.
5. like any described DNA store method of claim 1-4, comprise step:
(1) extracts DNA;
(2) DNA being dissolved in DNA as claimed in claim 1 preserves in the solvent and preserves.
6. DNA store method as claimed in claim 5 is characterized in that: said storage temperature is-20 ℃～-196 ℃.
7. DNA store method as claimed in claim 5 is characterized in that: the ultimate density that said DNA preserves is 1～10 μ g/L.
8. DNA store method as claimed in claim 7 is characterized in that: the ultimate density that said DNA preserves is 2～3 μ g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104032005A (en) * | 2014-06-12 | 2014-09-10 | 成都中创清科医学检验所有限公司 | Preservative and preservation method for intermediate PCR (Polymerase Chain Reaction) sequencing product |
| CN108330127A (en) * | 2018-03-09 | 2018-07-27 | 佛山市优特医疗科技有限公司 | A kind of magnetic bead mixed liquor, nucleic acid preservation method, nucleic acid extracting reagent and kit |
| CN111733156A (en) * | 2020-06-24 | 2020-10-02 | 北京启衡星生物科技有限公司 | Low-concentration DNA protective agent |
-
2010
- 2010-12-17 CN CN2010105950553A patent/CN102559654A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104032005A (en) * | 2014-06-12 | 2014-09-10 | 成都中创清科医学检验所有限公司 | Preservative and preservation method for intermediate PCR (Polymerase Chain Reaction) sequencing product |
| CN104032005B (en) * | 2014-06-12 | 2015-11-25 | 成都中创清科医学检验所有限公司 | A kind of preservatives of PCR order-checking intermediate product and store method |
| CN108330127A (en) * | 2018-03-09 | 2018-07-27 | 佛山市优特医疗科技有限公司 | A kind of magnetic bead mixed liquor, nucleic acid preservation method, nucleic acid extracting reagent and kit |
| CN108330127B (en) * | 2018-03-09 | 2021-08-17 | 佛山市优特医疗科技有限公司 | Magnetic bead mixed solution, nucleic acid storage method, nucleic acid extraction reagent and kit |
| CN111733156A (en) * | 2020-06-24 | 2020-10-02 | 北京启衡星生物科技有限公司 | Low-concentration DNA protective agent |
| CN111733156B (en) * | 2020-06-24 | 2021-12-14 | 北京启衡星生物科技有限公司 | Low-concentration DNA protective agent |
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Application publication date: 20120711 |