CN102747069A - Non-invasive extraction method of Haliotis discus hannai ino genome DNA - Google Patents
Non-invasive extraction method of Haliotis discus hannai ino genome DNA Download PDFInfo
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- CN102747069A CN102747069A CN2012102566715A CN201210256671A CN102747069A CN 102747069 A CN102747069 A CN 102747069A CN 2012102566715 A CN2012102566715 A CN 2012102566715A CN 201210256671 A CN201210256671 A CN 201210256671A CN 102747069 A CN102747069 A CN 102747069A
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Abstract
The invention discloses a non-invasive extraction method of Haliotis discus hannai ino genome DNA, comprising the steps of using 75% of alcohol to sterilize and irritate pallium of Haliotis discus hannai ino; scraping to obtain a mixture of mucus and epidermal tissue fragments; treating the mixture, centrifugally separating the mixture to obtain precipitate; adding a cell lysate and protease K to the precipitate; after digesting and oscillating, cooling the mixture for a certain time; adding NH4Ac to the sample; centrifugally separating to collect supernate; adding isopropanol to the supernate; after oscillating and mixing uniformly, and then centrifugally separating to obtain precipitate; using ethanol to wash the precipitate; drying the precipitate at room temperature for 5 minutes to obtain the Haliotis discus hannai ino genome DNA. The non-invasive extraction method of the Haliotis discus hannai ino genome DNA is simple, efficient and convenient, does not damage the Haliotis discus hannai ino and does not influence survival ability and production performance.
Description
Technical field
The present invention relates to a kind of process for extracting of genomic dna, particularly a kind of not damaged process for extracting of haliotis discus hannai Ino genomic dna.
Background technology
Haliotis discus hannai Ino (Haliotisdiscus hannai) belongs to Mollusca Gastropoda, Probranchia, Archaeogastropoda, is unique kind that the Bao Ke shellfish is distributed in northern China, also is the important object of Liaoning and Shandong coastal waters sea farming.Utilize modern molecular biology technique, haliotis discus hannai Ino is carried out operations such as population genetic structural analysis and genetic improvement, all must the genomic dna of haliotis discus hannai Ino be extracted.Traditional animal gene group process for extracting mainly is to be the DNA extraction object with muscle tissue or PBC; These methods all can cause haliotis discus hannai Ino to be in stress situation inevitably; Cause permanent damage even death, can't make subjects continue to be applied to produce.At present; International shellfish breeding has come into vogue based on the Walk-back back-and-forth method of paternity identification; And adopt this kind method before selection, the family ownership to the filial generation shellfish to identify; Therefore need now to adopt a kind of undamaged DNA extraction method, in order to avoid influence survival ability and the production performance of Bao.
Summary of the invention
The present invention is in order to solve the above-mentioned deficiency of existing in prior technology, proposes a kind of simply, efficient, convenient, and can not cause damage to haliotis discus hannai Ino, does not influence the haliotis discus hannai Ino genomic dna not damaged process for extracting of its survival ability and production performance.
Technical solution of the present invention is: a kind of not damaged process for extracting of haliotis discus hannai Ino genomic dna is characterized in that may further comprise the steps:
A, use the cotton balls speckle with 70%-75% alcohol that the mantle of haliotis discus hannai Ino is carried out disinfection and stimulate; The haliotis discus hannai Ino outside of belly after handling is left standstill up, treat secreting mucus after, scrape the mixture of getting 0.1-0.2g mucus and face tissue's fragment; Fix adding absolute ethyl alcohol in this mixture; After leaving standstill 5-10 minute on ice, deposition is got in spinning;
Add cell pyrolysis liquid in b, the gained deposition, the concussion mixing;
Adding 6 μ l-20 μ l in the biased sample of c, acquisition in the last step, the Proteinase K of 5mg/ml-10mg/ml, digestion is 3-5 hour under 65 ℃ condition, and concussion in per 15 minutes is once, under 4 ℃ of conditions, cools off 10-20 minute after the digestion completion;
D, in cooled sample, add the NH of 7.5M/L
4Ac, the volume of adding are that population of samples is long-pending
, leave standstill 5-10 minute on ice after, centrifugal under 4 ℃ of conditions, get supernatant;
E, in the supernatant of gained, add the Virahol identical, fully shake mixing with its volume, leave standstill 2-5 minute on ice after, centrifuging and taking precipitates;
F, gained precipitate the washing with alcohol twice with 70%-75%, shine under the room temperature and put 5-10 minute, obtain the haliotis discus hannai Ino genomic dna.
The present invention compares with prior art, has following advantage:
The key distinction of the present invention and traditional animal gene group DNA extraction method is; Ethanol through 75% stimulates the mantle of haliotis discus hannai Ino; Make its secreting mucus, and promptly hang the face tissue's fragment that together obtains when getting mucus and then extract genomic dna through handling mucus.The advantage of present method is: at first, protected the individual tissue integrity of sampling, do not caused any mechanical injuries, can from stress situation, recover through not long decubation individuality, can repeatedly take a sample for a long time; Secondly, clean, played disinfection and reduced the effect of foreign DNA interferential, help obtaining high-quality DNA through 75% ethanol.Present method gained DNA can be widely used in the aspects such as Genetic Constitution of Population analysis, phyletic evolution and molecular mark of haliotis discus hannai Ino, also for to utilize the situation of growing of molecule means continuous monitoring particular individual to lay a good foundation.Present method can also be applied in the breeding of other shellfish such as haliotis diversicolor Reeve, chlamys farreri etc. simultaneously.Therefore we can say that this DNA extraction method has possessed multiple advantage, be particularly suitable for applying in the art that its market and scientific research prospect are very wide.
Embodiment
Embodiment of the present invention is described below.
Embodiment one
The not damaged process for extracting of haliotis discus hannai Ino genomic dna, it may further comprise the steps: at first get fresh and alive haliotis discus hannai Ino, wash repeatedly 3 times with aseptic artificial seawater, make its outside of belly up, left standstill under the room temperature 5 minutes.The cotton balls that use speckles with 70% alcohol carries out disinfection to the mantle of haliotis discus hannai Ino and stimulates, and the haliotis discus hannai Ino outside of belly after handling was left standstill 5 minutes up, treat secreting mucus after; Scrape the mixture of getting 0.1g mucus and face tissue's fragment; And mixture put into centrifuge tube, and in centrifuge tube, adding 600 microlitre absolute ethyl alcohols and carry out sample and fix, the sample after fixing is after leaving standstill 8 minutes on ice; Last whizzer separates, taking precipitate;
In the throw out of gained, add cell pyrolysis liquid 600 microlitres, the cell pyrolysis liquid is here recommended to select Tris-Cl 100 mM for use, EDTA 50 mM, and 1% SDS, pH 8.0, and the concussion mixing;
The Proteinase K (its concentration is 10mg/ml) of adding 6 μ l in the sample behind the mixing, and under 65 ℃ of conditions, digested 3 hours, concussion in per 15 minutes was once cooled off 10 minutes under 4 ℃ of conditions after digestion is accomplished;
The NH that in cooled sample, adds 7.5M/L
4Ac, the volume of adding be population of samples long-pending 1/3rd, leave standstill 5 minutes on ice after, centrifugal under 4 ℃ of conditions, get supernatant;
In the supernatant that obtains, add isopyknic Virahol, fully shake mixing, leave standstill 2 minutes on ice after, the centrifuging and taking deposition;
The deposition of gained with 70% washing with alcohol twice, and is at room temperature shone and put 5 minutes, just obtain the haliotis discus hannai Ino genomic dna; Genomic dna is kept in the absolute ethyl alcohol under 4 ℃ condition, or is dissolved in subsequent use getting final product in an amount of sterilized water.
Embodiment two
The not damaged process for extracting of haliotis discus hannai Ino genomic dna, it may further comprise the steps: at first get fresh and alive haliotis discus hannai Ino, wash repeatedly 3 times with aseptic artificial seawater, make its outside of belly up, left standstill under the room temperature 5 minutes.The cotton balls that use speckles with 72% alcohol carries out disinfection to the mantle of haliotis discus hannai Ino and stimulates, and the haliotis discus hannai Ino outside of belly after handling was left standstill 5 minutes up, treat secreting mucus after; Scrape the mixture of getting 0.15g mucus and face tissue's fragment; And mixture put into centrifuge tube, and in centrifuge tube, adding 600 microlitre absolute ethyl alcohols and carry out sample and fix, the sample after fixing is after leaving standstill 5 minutes on ice; Last whizzer separates, taking precipitate;
In the throw out of gained, add cell pyrolysis liquid 600 microlitres, the cell pyrolysis liquid is here recommended to select Tris-Cl 100 mM for use, EDTA 50 mM, and 1% SDS, pH 8.0, and the concussion mixing;
The Proteinase K (its concentration is 8mg/ml) of adding 12 μ l in the sample behind the mixing, and under 65 ℃ of conditions, digested 4 hours, concussion in per 15 minutes was once cooled off 15 minutes under 4 ℃ of conditions after digestion is accomplished;
The NH that in cooled sample, adds 7.5M/L
4Ac, the volume of adding be population of samples long-pending 1/3rd, leave standstill 8 minutes on ice after, centrifugal under 4 ℃ of conditions, get supernatant;
In the supernatant that obtains, add isopyknic Virahol, fully shake mixing, leave standstill 3 minutes on ice after, the centrifuging and taking deposition;
The deposition of gained with 73% washing with alcohol twice, and is at room temperature shone and put 8 minutes, just obtain the haliotis discus hannai Ino genomic dna; Genomic dna is kept in the absolute ethyl alcohol under 4 ℃ condition, or is dissolved in subsequent use getting final product in an amount of sterilized water.
Embodiment three
The not damaged process for extracting of haliotis discus hannai Ino genomic dna, it may further comprise the steps: at first get fresh and alive haliotis discus hannai Ino, wash repeatedly 3 times with aseptic artificial seawater, make its outside of belly up, left standstill under the room temperature 5 minutes.The cotton balls that use speckles with 75% alcohol carries out disinfection to the mantle of haliotis discus hannai Ino and stimulates, and the haliotis discus hannai Ino outside of belly after handling was left standstill 5 minutes up, treat secreting mucus after; Scrape the mixture of getting 0.2g mucus and face tissue's fragment; And mixture put into centrifuge tube, and in centrifuge tube, adding 600 microlitre absolute ethyl alcohols and carry out sample and fix, the sample after fixing is after leaving standstill 10 minutes on ice; Last whizzer separates, taking precipitate;
In the throw out of gained, add cell pyrolysis liquid 600 microlitres, the cell pyrolysis liquid is here recommended to select Tris-Cl 100 mM for use, EDTA 50 mM, and 1% SDS, pH 8.0, and the concussion mixing;
The Proteinase K (its concentration is 5mg/ml) of adding 20 μ l in the sample behind the mixing, and under 65 ℃ of conditions, digested 5 hours, concussion in per 15 minutes was once cooled off 20 minutes under 4 ℃ of conditions after digestion is accomplished;
The NH that in cooled sample, adds 7.5M/L
4Ac, the volume of adding be population of samples long-pending 1/3rd, leave standstill 10 minutes on ice after, centrifugal under 4 ℃ of conditions, get supernatant;
In the supernatant that obtains, add isopyknic Virahol, fully shake mixing, leave standstill 5 minutes on ice after, the centrifuging and taking deposition;
The deposition of gained with 75% washing with alcohol twice, and is at room temperature shone and put 10 minutes, just obtain the haliotis discus hannai Ino genomic dna; Genomic dna is kept in the absolute ethyl alcohol under 4 ℃ condition, or is dissolved in subsequent use getting final product in an amount of sterilized water.
Claims (1)
1. the not damaged process for extracting of a haliotis discus hannai Ino genomic dna is characterized in that may further comprise the steps:
A, use the cotton balls speckle with 70%-75% alcohol that the mantle of haliotis discus hannai Ino is carried out disinfection and stimulate; The haliotis discus hannai Ino outside of belly after handling is left standstill up, treat secreting mucus after, scrape the mixture of getting 0.1-0.2g mucus and face tissue's fragment; Fix adding absolute ethyl alcohol in this mixture; After leaving standstill 5-10 minute on ice, deposition is got in spinning;
Add cell pyrolysis liquid in b, the gained deposition, the concussion mixing;
Adding 6 μ l-20 μ l in the biased sample of c, acquisition in the last step, the Proteinase K of 5mg/ml-10mg/ml, digestion is 3-5 hour under 65 ℃ condition, and concussion in per 15 minutes is once, under 4 ℃ of conditions, cools off 10-20 minute after the digestion completion;
D, in cooled sample, add the NH of 7.5M/L
4Ac, the volume of adding are that population of samples is long-pending
, leave standstill 5-10 minute on ice after, centrifugal under 4 ℃ of conditions, get supernatant;
E, in the supernatant of gained, add the Virahol identical, fully shake mixing with its volume, leave standstill 2-5 minute on ice after, centrifuging and taking precipitates;
F, gained precipitate the washing with alcohol twice with 70%-75%, shine under the room temperature and put 5-10 minute, obtain the haliotis discus hannai Ino genomic dna.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184215A (en) * | 2013-04-10 | 2013-07-03 | 中国水产科学研究院长江水产研究所 | Method for extracting genomic DNA from giant salamander skin mucus |
| CN103540586A (en) * | 2013-10-08 | 2014-01-29 | 威海长青海洋科技股份有限公司 | Extraction method of haliotis discus hannai ino high-purity genomic deoxyribonucleic acid (DNA) sample |
| CN104164418A (en) * | 2014-06-26 | 2014-11-26 | 北京圣谷同创科技发展有限公司 | Paraffin-embedded tissue slice genome DNA extraction kit and using method thereof |
| CN104975001A (en) * | 2015-07-15 | 2015-10-14 | 中国海洋大学 | Method for nondestructive extraction of rapana venosa genome DNA |
| CN105368819A (en) * | 2015-12-17 | 2016-03-02 | 中国水产科学研究院淡水渔业研究中心 | Method for extracting genome DNA from ricefield eel body surface mucus |
| CN105400775A (en) * | 2015-12-31 | 2016-03-16 | 中国长江三峡集团公司中华鲟研究所 | Method for harmlessly extracting Chinese sturgeon DNA |
| CN106701744A (en) * | 2017-02-09 | 2017-05-24 | 中国科学院南海海洋研究所 | Method for living-body extraction of tridacna crocea genomic DNA |
| CN108251416A (en) * | 2018-04-26 | 2018-07-06 | 海南归耘田农业科技有限公司 | A kind of method of not damaged extraction Procambius clarkii DNA |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184215A (en) * | 2013-04-10 | 2013-07-03 | 中国水产科学研究院长江水产研究所 | Method for extracting genomic DNA from giant salamander skin mucus |
| CN103540586A (en) * | 2013-10-08 | 2014-01-29 | 威海长青海洋科技股份有限公司 | Extraction method of haliotis discus hannai ino high-purity genomic deoxyribonucleic acid (DNA) sample |
| CN104164418A (en) * | 2014-06-26 | 2014-11-26 | 北京圣谷同创科技发展有限公司 | Paraffin-embedded tissue slice genome DNA extraction kit and using method thereof |
| CN104975001A (en) * | 2015-07-15 | 2015-10-14 | 中国海洋大学 | Method for nondestructive extraction of rapana venosa genome DNA |
| CN105368819A (en) * | 2015-12-17 | 2016-03-02 | 中国水产科学研究院淡水渔业研究中心 | Method for extracting genome DNA from ricefield eel body surface mucus |
| CN105400775A (en) * | 2015-12-31 | 2016-03-16 | 中国长江三峡集团公司中华鲟研究所 | Method for harmlessly extracting Chinese sturgeon DNA |
| CN106701744A (en) * | 2017-02-09 | 2017-05-24 | 中国科学院南海海洋研究所 | Method for living-body extraction of tridacna crocea genomic DNA |
| CN108251416A (en) * | 2018-04-26 | 2018-07-06 | 海南归耘田农业科技有限公司 | A kind of method of not damaged extraction Procambius clarkii DNA |
| CN108251416B (en) * | 2018-04-26 | 2023-09-15 | 海南归耘田农业科技有限公司 | Method for extracting procambarus clarkia DNA without damage |
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