CN108004232B - Human genome DNA specimen is stored at room temperature method - Google Patents
Human genome DNA specimen is stored at room temperature method Download PDFInfo
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- CN108004232B CN108004232B CN201711276915.5A CN201711276915A CN108004232B CN 108004232 B CN108004232 B CN 108004232B CN 201711276915 A CN201711276915 A CN 201711276915A CN 108004232 B CN108004232 B CN 108004232B
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- 229920000936 Agarose Polymers 0.000 claims abstract description 26
- 238000001035 drying Methods 0.000 claims abstract description 10
- 238000002844 melting Methods 0.000 claims abstract description 6
- 230000008018 melting Effects 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 65
- 238000004321 preservation Methods 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 6
- 210000002919 epithelial cell Anatomy 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004209 hair Anatomy 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 210000000214 mouth Anatomy 0.000 description 4
- 210000002200 mouth mucosa Anatomy 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
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- 239000000499 gel Substances 0.000 description 3
- 210000003780 hair follicle Anatomy 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000003196 chaotropic effect Effects 0.000 description 2
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- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 210000003296 saliva Anatomy 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
- 208000028782 Hereditary disease Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108091093078 Pyrimidine dimer Proteins 0.000 description 1
- ASJWEHCPLGMOJE-LJMGSBPFSA-N ac1l3rvh Chemical compound N1C(=O)NC(=O)[C@@]2(C)[C@@]3(C)C(=O)NC(=O)N[C@H]3[C@H]21 ASJWEHCPLGMOJE-LJMGSBPFSA-N 0.000 description 1
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- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 238000006392 deoxygenation reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 230000005496 eutectics Effects 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- XUXNAKZDHHEHPC-UHFFFAOYSA-M sodium bromate Chemical compound [Na+].[O-]Br(=O)=O XUXNAKZDHHEHPC-UHFFFAOYSA-M 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
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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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- Wood Science & Technology (AREA)
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Abstract
A kind of human genome DNA specimen is stored at room temperature method, the human genome DNA solution that preparation is extracted is mixed with the low melting-point agarose solution in melting state, the powdered sample of human genome DNA that can be stored at room temperature is prepared after drying, the powdered sample is fitted into container and is sealed, then is placed in light resistant container or environment.The present invention is suitable for being stored at room temperature for human body base group DNA sample.
Description
Technical field
The present invention relates to the methods that is stored at room temperature of cdna sample, the especially side of being stored at room temperature of human genome DNA specimen
Method.
Background technique
Human genome DNA is all DNA for forming human genome, and follow-on whole can be passed to from a generation by referring to
Hereditary information.Human genome DNA contains the hereditary information of human body whole, is heredity and the material base that species continue, it makes
There is character similar with parental generation in offspring.Saving human genome DNA specimen is the whole life hereditary information for saving human body.With
Being constantly progressive for the Protocols in Molecular Biologies such as gene order-checking, scientist can hereditary information and life to genomic DNA
Password is analyzed and is interpreted, human genome DNA specimen by and various gene clonings, disease will be applied more broadly in
Diagnosis, medication guide, individual identification, paternity identification, hereditary disease parsing, gene family tree building and preservation etc..
A kind of DNA molecular long-term preservation and visual storing method and its device（Application number 200810017680.2）Institute
It is come with some shortcomings with method, such as：（1）In conjunction with the nucleic acid dyes such as DNA molecular and EB, and often there is carcinogenic equal life in the dyestuffs such as EB
Object toxicity；（2）It need to tap rubber under ultraviolet light to target DNA molecule, since ultraviolet light can cause the damage of DNA molecular, energy
The formation of thymine dimer and the crosslinking of DNA chain are induced, the distortion of DNA molecular information is caused, loses DNA molecular preservation
Value and significance；（3）It needs to separate DNA molecular by electrophoresis, electrophoresis process is potential to generate damage to DNA molecular.
For the stability of the integrality of holding human genome DNA gene structure, physicochemical property and biological activity, carry out base both at home and abroad
Because the genomic DNA sample being prepared usually is stored under condition of ultralow temperature by the company of Sample preservation service or mechanism, keep away
Exempt from physics, chemistry and biological factor to have an adverse effect to human genome DNA specimen.However, this cryopreservation strategy
Also many drawbacks are brought, as expense is very expensive, need professional equipment, individual to be difficult to carry out the problems such as.
Summary of the invention
The invention solves implementation conditions existing during Regular Human's genomic DNA Sample preservation, and harsh, expense is held high
It is expensive, need professional equipment, storage need to be pinpointed, individual is difficult to the problems such as saving, human genome DNA of the invention is provided thus
Sample is stored at room temperature method, and this method operates fairly simple, cost saving, and Sample preservation is easy, and can protect for a long time at room temperature
It deposits.
Human genome DNA specimen of the present invention is stored at room temperature method, including by human genome DNA solution and locates
It is mixed in the low melting-point agarose solution of melting state, the human genome DNA powder that can be stored at room temperature is prepared after drying
The powdered sample is fitted into container and is sealed, then is placed in light resistant container or environment by last shape sample.
The powdered DNA sample, which is packed into container and is sealed, can refer to powdered DNA sample loading vial
It is sealed after nitrogen charging deoxygenation, to be more advantageous to long-term preservation of human genome DNA's sample under normal temperature environment.
The low melting-point agarose solution is suspended in low melting-point agarose in the buffers such as water or TE, due to eutectic
The fusing point of point agarose is not higher than 65 DEG C, therefore, can form low melting-point agarose solution when being not higher than 65 DEG C.What is melted is low
About 26-30 DEG C of its gel point of melt agarose sugar juice, do not influence human genome DNA structure and bioactivity at a temperature of,
Human genome DNA specimen is added in low melting-point agarose solution, mixing well makes DNA sample be dispersed in low melting point fine jade
In lipolysaccharide.
Drying of the present invention can be natural drying or low-temperature vacuum drying in air at room temperature.
The carrying genes group DNA in low melting-point agarose powder, only need rehydration and by recovery to solution state can release
Genomic DNA is put, can be directly used for the molecular Biological Detections such as genetic analysis after recycling genomic DNA.
The present invention due to by DNA solution in melting state low melting-point agarose solution mix, make DNA wrapped up into
In low melting-point agarose, the powdered sample of load DNA is made after drying, therefore the present invention is simple for production, expense is low, institute's sample preparation
This preservation is convenient, and is easy to make subsequent analysis to genomic DNA sample.Low melting-point agarose is safe and stable inert component,
Also be the good carrier of DNA, the powder form formed after drying have the advantages that it is a variety of, such as（1）Genomic DNA sample package
In low melting-point agarose powder, the direct collision between DNA sample is avoided, protect the stability of DNA sample structure and complete
Property.（2）The sharp impacts between powder can be greatly reduced in powder form, ensure the stability and integrality of DNA sample structure.
（3）Human genome DNA specimen can be dispensed on demand, be taken on demand.（4）It is mentioned again from low melting-point agarose powder
When taking human genome DNA, only need rehydration and by recovery to the i.e. releasable genomic DNA of solution state.
The present invention since the described powdered sample comprising DNA is packed into container and is sealed, sample seal up for safekeeping it is more convenient,
And long-term preservation, especially container can more have except being sealed after oxygen and filling nitrogen to the long-term preservation of DNA at room temperature at room temperature
Benefit.
Specific embodiment
1. human oral cavity mucomembranous epithelial cell of embodiment prepares genomic DNA sample and seals up for safekeeping
1, the acquisition of human oral cavity mucomembranous epithelial cell
It is first gargled for several times with clear water before sampling, then is scraped repeatedly in oral cavity wall repeatedly, so that oral cavity is wiped with buccal swab
Oral mucosa epithelial cell is carried on son.
2, lytic cell
The material for carrying oral mucosa epithelial cell is cut, Eppendorf is placed in（EP）Guan Zhong adds into EP pipe
The cell pyrolysis liquid for entering 400 μ L is mixed well to without apparent cell mass.Cell cracking formula of liquid is：10 mmol/L
Tris-HCl (pH8.0), 30 mmol/L EDTA (pH8.0), 0.5% SDS, RNase A (20 μ g/mL).
3, chaotropic agent processing release genomic DNA
The chaotropic agent sodium bromate of final concentration of 1 mol/L is added into EP pipe, mixes well.
4, isolating protein is removed
Isometric phenol/chloroform/isoamyl alcohol (25 is added into the EP pipe of step (3):24:1), oscillation mixes, and centrifugation is extremely
Solution layering, supernatant liquid is transferred in the EP pipe of another cleaning.It is primary to repeat this step, and with chloroform it is primary on
Clearly, the supernatant containing genomic DNA is transferred in another cleaning EP pipe.
5, DNA is precipitated
The dehydrated alcohol of 2.5 times of volumes is added into the EP pipe of step (4), genomic DNA is collected by centrifugation, adds into precipitating
The 70% ethanol washing DNA for entering 1 mL, removes supernatant, and opening dries genome, the human genome DNA specimen as precipitated.
6, the dissolution of DNA
The distilled water or TE buffer of 100 μ L, dissolution genomic DNA precipitating are added into the EP pipe of step (5).It utilizes
Light densitometry measures the concentration of genomic DNA, utilizes the integrality of agarose gel electrophoresis detection genomic DNA.
7, the configuration of low melting-point agarose solution
Low melting-point agarose distilled water or TE buffer are suspended, being heated to 65 DEG C or so melts low melting-point agarose
The agarose solution for being 0.8-2% as mass fraction.
8, genome DNA sample is dispersed in low melting-point agarose solution
By step（6）Obtained human genome DNA specimen is added to step（7）Preparation, also in the low of molten state
It in melt agarose sugar juice, is mixed by inversion, forms gel after standing, after gel drying, vacuum drying or low-temperature vacuum drying
Obtain white powder solid, the human genome DNA specimen that can be as stored at room temperature.
9, it seals up for safekeeping
By step（8）Made sample is transferred in glass container, nitrogen charging deoxidation, under nitrogen environment at one atm
It is sealed, then is placed in light resistant container or environment.
Embodiment 2. prepares genomic DNA sample with blood of human body and its seals up for safekeeping
1, blood of human body sample acquires
Acquisition anticoagulation 0.5-2ml prepares blood cake.This blood sample in -80 DEG C of stored frozens, can at least maintain 2 years without
It is rotten.
2, splitting erythrocyte
The erythrocyte cracked liquid of 400 μ L is added into the anticoagulation of 200 μ L, 2000g is centrifuged 10 min after mixing, abandons
Supernatant.If still there is more red blood cell residual, it is primary also to repeat cracking.
The step of subsequent operation and embodiment 1（2）-（9）Unanimously.
Embodiment 3. prepares genomic DNA sample with human body hair follicle and its seals up for safekeeping
It is chosen 3, hair with clean tweezers, wash with distilled water the attachment of hair and root hair follicle, then from hair
Root of hair hair follicle part is immersed in the centrifuge tube containing 400 μ l cell pyrolysis liquids, is gone after cell cracking is abundant by root clip 1cm
The step of hair removal and other impurity, subsequent operation and embodiment 1（2）-（9）Unanimously.
Embodiment 4. prepares genomic DNA sample with saliva and its seals up for safekeeping
2000 g of saliva is centrifuged 10 min, abandons supernatant, with the physiological saline suspension oral mucosa epithelial cell of 1 mL,
2000 g are centrifuged 10 min again, abandon supernatant, and precipitating is oral mucosa epithelial cell.The step of subsequent operation and embodiment 1
（2）-（9）Unanimously.
Claims (3)
1. human genome DNA specimen is stored at room temperature method, which is characterized in that including by human genome DNA solution and place
It is mixed in the low melting-point agarose solution of melting state, the human genome DNA powder that can be stored at room temperature is prepared after drying
The powdered sample is fitted into container and is sealed by last shape sample；
The human genome DNA solution is the solution that human genome is dissolved in that distilled water or TE buffer are formed；
The configuration method of the low melting-point agarose solution is：Low melting-point agarose distilled water or TE buffer are suspended, added
The low melting-point agarose solution that heat makes low melting-point agarose be melted into mass fraction 0.8-2% to 65 DEG C；
The drying is drying, vacuum drying；
The powdered sample be fitted into container and seal refer to by powdered sample be packed into container through remove oxygen and filling nitrogen after seal.
2. being stored at room temperature method as described in claim 1, which is characterized in that be directly dispersing in Genomic DNA solution and be in
In the low melting-point agarose solution of melting state.
3. being stored at room temperature method as described in claim 1, which is characterized in that the powdered sample to be fitted into container and seal
Mouthful, then be placed in light resistant container or environment.
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| CN113862332A (en) * | 2021-09-17 | 2021-12-31 | 浙江大学 | Application of agarose in preparation of biomacromolecule freeze-drying protective agent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1288956A (en) * | 2000-09-13 | 2001-03-28 | 安徽省农业科学院 | Preservation method of mixed liquid of polymerase chair reaction |
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| CN102703429B (en) * | 2012-06-01 | 2013-09-18 | 中国水产科学研究院黄海水产研究所 | Nucleic acid isothermal amplification reaction reagent suitable for being stored and transported at normal temperature |
| CN102703428B (en) * | 2012-06-01 | 2013-09-18 | 中国水产科学研究院黄海水产研究所 | Gel-based preservation method for polymerase chain reaction agent and reaction reagent |
| CN105463125A (en) * | 2016-02-02 | 2016-04-06 | 江苏正大天创生物工程有限公司 | Nucleic acid amplification system and freeze-drying protective agent thereof |
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| CN1288956A (en) * | 2000-09-13 | 2001-03-28 | 安徽省农业科学院 | Preservation method of mixed liquid of polymerase chair reaction |
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