CN103243089B - Method for extracting deoxyribonucleic acid (DNA) from tanned animal specimen hide - Google Patents
Method for extracting deoxyribonucleic acid (DNA) from tanned animal specimen hide Download PDFInfo
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- CN103243089B CN103243089B CN201310196655.6A CN201310196655A CN103243089B CN 103243089 B CN103243089 B CN 103243089B CN 201310196655 A CN201310196655 A CN 201310196655A CN 103243089 B CN103243089 B CN 103243089B
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Abstract
The invention relates to a method for extracting deoxyribonucleic acid (DNA) from a tanned animal specimen hide. The DNA extracting quantity is small, heavy metal ions exist, and the polymerase chain reaction (PCR) amplification result is low, so the conventional method is not suitable for molecular biological research. The method comprises the following steps of: soaking the specimen hide in alcohol at normal temperature, washing with distilled water until no sundries exist, treating by using a Na2HPO4 solution for 20 to 28 hours under the condition of continuously oscillating, and washing with distilled water for 2 to 3 times; soaking a specimen in a soaking solution for 1.5 to 2.5 hours, placing the specimen into a centrifugal tube, adding 500mu l of digestive solution into the centrifugal tube, performing centrifugal separation and precipitating; and adding 7mu l of completely suspended DNAIQTM resin of a DNAIQTM system during secondary extraction, performing vortex oscillation at a high speed for many seconds, performing magnetic separation, and transferring the DNA solution to a container carefully after separation is finished. According to the method, the heavy metal ions can be effectively removed, the product has high concentration and high purity, and the method is suitable for all the tanned specimen hides.
Description
Technical field
The present invention relates to biological DNA technique field, be specifically related to the extracting method of a kind of tanned animal sample hide DNA.
Background technology
Along with the development of science and technology, DNA molecular marker technology has been widely used in biological genome research, all many-sides such as biological genetic breeding, origin evolution, classification, medical science and legal medical expert solve a case, and become one of main flow of modern molecular genetics and molecular biology research and application.These are all the research for DNA, so extract high purity, the DNA of high density is the basis of all research.For the extraction of animal DNA, modal is obtain from muscle, blood and various internal organ, because the DNA content of these tissues is many, organized processing is easier to, and the DNA fragmentation of proposition is more complete, is applicable to various molecular biology research.But the molecule genetics research of wildlife is often subject to the restriction of spatial distribution, sample collecting time span etc. on individual amount, sample source ground.Obtain the muscle of some animals, blood, or the experiment material such as internal organ is all very difficult, for some, oneself is through the animal of extinction, and this is exactly impossible at all.Therefore, during conducting molecule biological study, collection tanned animal sample is needed to carry out the deficiency of supplementary field specimen collection.
The extracting method of what the extraction of current tanned animal sample hide DNA adopted is conventional animal DNA, but facts have proved that from sample, to extract DNA infeasible often.Because these samples are mostly all through various process, containing enzyme inhibitorss etc. such as a large amount of water-soluble heavy metal ion, tanning agents, interference is to a certain degree had to DNA extraction, the DNA extracted from these samples is caused to measure general all fewer, when carrying out pcr amplification and checking DNA extraction, only have and can amplify band less than 10%, DNA is extracted in display.Therefore prior art Problems existing is: DNA extraction amount is little and have heavy metal ion, and pcr amplification result is low, is not suitable for and carries out molecular biology research.
summary of the invention:
The object of the invention is the extracting method that will provide a kind of efficient tanned animal sample hide DNA, little and have heavy metal ion to overcome the DNA extraction amount that prior art exists, pcr amplification result is low, is not suitable for the problem of carrying out molecular biology research.
The object of the present invention is achieved like this, and the extracting method of a kind of tanned animal sample hide DNA, comprises the steps: successively
1) process of raw material:
1. by the zoological specimens hide through tanning process at normal temperatures with alcohol-pickled, more for subsequent use to no-sundries with distilled water flushing;
2. clip hide, uses Na
2hPO
4solution processes 20 ~ 28 hours under continuous oscillating condition, with distilled water flushing 2 ~ 3 times;
2) digest:
Soaked 1.5 ~ 2.5 hours by sample soak solution, abandon soak solution, shred with scissors as far as possible, be placed in centrifuge tube, add 500 μ l Digestive systems in centrifuge tube, 24ul 0.5mol/L EDTA, 6ul 20mg/ml Proteinase K, deposits 3-5 hour at 55 DEG C; Centrifugation, gets 200 μ l supernatant liquors in another centrifuge tube;
Described soak solution is 10mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L NaCl, pH8.0;
3) be separated, precipitate:
In centrifuge tube, add the saturated phenol of 200 μ lTris-(pH8.0), shake mixing 8 ~ 12 minutes lightly, centrifugation, repeats this step once; Moved to by supernatant in a new centrifuge tube, then add 200 μ l chloroforms, shake mixing 8 ~ 12 minutes lightly, centrifugation, gets supernatant and repeats this step once; Get supernatant to move in a new centrifuge tube, add the cold dehydrated alcohol of 2.5 times of volumes, shake a moment lightly, be placed in refrigerator under-20 DEG C of conditions freezing 25 ~ 35 minutes; Centrifugation, careful supernatant discarded; Draw 200 μ L70% washing with alcohol for a moment, centrifugation, careful supernatant discarded, opens centrifuge tube lid, makes ethanol evaporation 10-20 minute under room temperature; By dissolving DNA precipitation under 30 μ l TE (10 mmol/L Tris-HCl, 10 mmol/L EDTA, pH8.0) damping fluid room temperature;
4) secondary extracting:
The DNA IQ that 7uL suspends completely is added in the DNA solution extracted
tMdNA IQ in system
tMresin, the high speed vortex oscillation several seconds, and carry out Magneto separate, be separated after terminating, DNA solution is transferred in container carefully.
Above-mentioned steps 2) in Digestive system be Promega company produce Nuclei Lysis Sol ' n reagent.
Above-mentioned steps 4) in Magneto separate step comprise the steps:
1. be placed on by pipe on Magneto separate frame, Magneto separate carries out at once.The all solution of careful removal, does not touch the resin on tube wall, adds 100 μ L lysis buffers, take off pipe from Magneto separate frame, and the high speed vortex oscillation several seconds, pipe is put back on Magneto separate frame, and removes all lysates;
2. add the 1x washings that 100 μ L prepare, take off pipe from Magneto separate frame, and the high speed vortex oscillation several seconds, pipe is put back on Magneto separate frame, removes all washingss; Uncap, is placed on Magneto separate frame, air drying by pipe, add 25-100 μ L elutriant, lid upper tube cap high speed vortex oscillation several seconds;
3. 65 DEG C of incubations 5 ~ 8 minutes, take out the pipe of incubation, the high speed vortex oscillation several seconds, are put on Magneto separate frame immediately, are separated and terminate.
Compared with prior art, advantage of the present invention is:
1, the present invention can effective heavy-metal ion removal, and product concentration is large, and purity is high, can be used for doing the experiment of pcr amplification equimolecular, and when carrying out pcr amplification and detecting DNA, have the sample of more than 85% to amplify band, DNA is extracted in display.
2, the present invention is applicable to all sample hides through tanning process, also can be used for the work such as true and false qualification, illegal trading wildlife goods species identification that the true and false qualification of leatherware, illegal trading wildlife goods species identification etc. also can be used for leatherware.
Embodiment:
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1:
An extracting method of tanned animal sample hide DNA, comprises the steps successively
1) process of raw material:
1. the woods musk deer sample hide through tanning process is used alcohol-pickled half an hour at normal temperatures, more for subsequent use to no-sundries with distilled water flushing;
2. clip about 0. 5 cm
2hide, with 1ml 0.03M Na
2hPO
4solution processes 24 hours under continuous oscillating condition, with distilled water flushing 3 times;
2) digest:
By sample soak solution (10mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L NaCl, pH8.0) soak 2 hours, abandon soak solution, shred with scissors as far as possible, be placed in 1. 5ml centrifuge tubes, in centrifuge tube, add 500 μ l Digestive systems (Nuclei Lysis Sol ' the n reagent that Promega company produces), 24ul 0.5mol/L EDTA, 6ul 20mg/ml Proteinase K, deposits 3 hours at 55 DEG C; Centrifugal 5 minutes of 12000r/min, gets 200 μ l supernatant liquors in another centrifuge tube;
3) be separated, precipitate:
In centrifuge tube, add the saturated phenol of 200 μ lTris-(pH8.0), lightly mixed 10 minutes of shake, centrifugal 10 minutes of 12000 r/min, repeat this step once; Moved to by supernatant in a new 1.5ml centrifuge tube, then add 200 μ l chloroforms, shake mixing 10 minutes lightly, centrifugal 10 minutes of 12000 r/min, get supernatant and repeat this step once; Get supernatant to move in a new 1.5ml centrifuge tube, add the cold dehydrated alcohol of 2.5 times of volumes, shake a moment lightly, be placed in refrigerator under-20 DEG C of conditions freezing 30 minutes; Centrifugal 15 min of 13000 r/min, careful supernatant discarded; Draw 200 μ L70% washing with alcohol for a moment, centrifugal 15 minutes of 13000 r/min, careful supernatant discarded, opens centrifuge tube lid under room temperature, make ethanol evaporation 20 minutes; By dissolving DNA precipitation under 30 μ l TE (10 mmol/L Tris-HCl, 10 mmol/L EDTA, pH8.0) damping fluid room temperature;
4) secondary extracting:
The DNA IQ that 7uL suspends completely is added in the DNA solution extracted
tMdNA IQ in system
tMresin, high speed vortex oscillation 3 second, and carry out Magneto separate, described Magneto separate step comprises the steps:
1. pipe is placed on MagneSphere
on Magneto separate frame, Magneto separate carries out at once, all solution of careful removal, do not touch the resin on tube wall, add 100 μ L lysis buffers, take off pipe from Magneto separate frame, and high speed vortex oscillation 2 second, pipe is put back on Magneto separate frame, and removes all lysates;
2. the 1x washings that 100 μ L prepare is added, pipe is taken off from Magneto separate frame, and high speed vortex oscillation 2 second, pipe is put back on Magneto separate frame, removes all washingss, uncap, pipe is placed on Magneto separate frame, air drying 5 minutes, adds 25 μ L elutriants, lid upper tube cap high speed vortex oscillation 2 second.
3. 65 DEG C of incubations 5 minutes.Take out the pipe of incubation, high speed vortex oscillation 2 second, be put into immediately on Magneto separate frame.
After separation terminates, DNA solution is transferred in selected container carefully.
Carry out pcr amplification and detect DNA, DNA is extracted in display.
Embodiment 2:
An extracting method of tanned animal sample hide DNA, comprises the steps successively
1) process of raw material:
1. by the goral sample hide through tanning process at normal temperatures, with alcohol-pickled 40 minutes, for subsequent use to no-sundries with distilled water flushing again;
2. clip about 1 cm
2hide, with 5ml 0.03M Na
2hPO
4solution processes 28 hours under continuous oscillating condition, with distilled water flushing 3 times;
2) digest:
By sample soak solution (10mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L NaCl, pH8.0) soak 2.5 hours, abandon soak solution, shred with scissors as far as possible, be placed in 1. 5ml centrifuge tubes, in centrifuge tube, add 500 μ l Digestive systems (Nuclei Lysis Sol ' the n reagent that Promega company produces), 24ul 0.5mol/L EDTA, 6ul 20mg/ml Proteinase K, deposits 5 hours at 55 DEG C; Centrifugal 6 minutes of 12000r/min, gets 200 μ l supernatant liquors in another centrifuge tube;
3) be separated, precipitate:
In centrifuge tube, add the saturated phenol of 200 μ lTris-(pH8.0), lightly mixed 10 minutes of shake, centrifugal 8 minutes of 12000 r/min, repeat this step once; Moved to by supernatant in a new 1.5ml centrifuge tube, then add 200 μ l chloroforms, shake mixing 8 minutes lightly, centrifugal 12 minutes of 12000 r/min, get supernatant and repeat this step once; Get supernatant to move in a new 1.5ml centrifuge tube, add the cold dehydrated alcohol of 2.5 times of volumes, shake a moment lightly, be placed in refrigerator under-20 DEG C of conditions freezing 35 minutes; The centrifugal 12min of 13000 r/min, careful supernatant discarded; Draw 200 μ L70% washing with alcohol for a moment, centrifugal 12 minutes of 13000 r/min, careful supernatant discarded, opens centrifuge tube lid under room temperature, make ethanol evaporation 15 minutes; By dissolving DNA precipitation under 30 μ l TE (10 mmol/L Tris-HCl, 10 mmol/L EDTA, pH8.0) damping fluid room temperature;
4) secondary extracting:
The DNA IQ that 7uL suspends completely is added in the DNA solution extracted
tMdNA IQ in system
tMresin, high speed vortex oscillation 2 second, and carry out Magneto separate, described Magneto separate step comprises the steps:
1. pipe is placed on MagneSphere
on Magneto separate frame, Magneto separate carries out at once, all solution of careful removal, touches the resin on tube wall.Add 100 μ L lysis buffers, take off pipe from Magneto separate frame, and high speed vortex oscillation 3 second, pipe is put back on Magneto separate frame, and removes all lysates;
2. the 1x washings that 100 μ L prepare is added, pipe is taken off from Magneto separate frame, and high speed vortex oscillation 3 second, pipe is put back on Magneto separate frame, removes all washingss, uncap, pipe is placed on Magneto separate frame, air drying 8 minutes, adds 100 μ L elutriants, lid upper tube cap high speed vortex oscillation 3 second;
3. 65 DEG C of incubations 8 minutes.Take out the pipe of incubation, high speed vortex oscillation 3 second, be put into immediately on Magneto separate frame.
After separation terminates, DNA solution is transferred in selected container carefully.
Carry out pcr amplification and detect DNA, DNA is extracted in display.
Embodiment 3:
An extracting method of tanned animal sample hide DNA, comprises the steps successively
1) process of raw material:
1. by the rhinopithecus roxellanae sample hide through tanning process at normal temperatures, with alcohol-pickled 25 minutes, for subsequent use to no-sundries with distilled water flushing again;
2. clip about 0.8 cm
2hide, with 3ml 0.03M Na
2hPO
4solution processes 24 hours under continuous oscillating condition, with distilled water flushing 2 times;
2) digest:
By sample soak solution (10mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L NaCl, pH8.0) soak 1.5 hours, abandon soak solution, shred with scissors as far as possible, be placed in 1. 5ml centrifuge tubes, in centrifuge tube, add 500 μ l Digestive systems (Nuclei Lysis Sol ' the n reagent that Promega company produces), 24ul 0.5mol/L EDTA, 6ul 20mg/ml Proteinase K, deposits 3 hours at 55 DEG C; Centrifugal 5 minutes of 12000r/min, gets 200 μ l supernatant liquors in another centrifuge tube;
3) be separated, precipitate:
In centrifuge tube, add the saturated phenol of 200 μ lTris-(pH8.0), lightly mixed 8 minutes of shake, centrifugal 8 minutes of 12000 r/min, repeat this step once; Moved to by supernatant in a new 1.5ml centrifuge tube, then add 200 μ l chloroforms, shake mixing 8 minutes lightly, centrifugal 12 minutes of 12000 r/min, get supernatant and repeat this step once; Get supernatant to move in a new 1.5ml centrifuge tube, add the cold dehydrated alcohol of 2.5 times of volumes, shake a moment lightly, be placed in refrigerator under-20 DEG C of conditions freezing 25 minutes; The centrifugal 12min of 13000 r/min, careful supernatant discarded; Draw 200 μ L70% washing with alcohol for a moment, centrifugal 12 minutes of 13000 r/min, careful supernatant discarded, opens centrifuge tube lid under room temperature, make ethanol evaporation 15 minutes; By dissolving DNA precipitation under 30 μ l TE (10 mmol/L Tris-HCl, 10 mmol/L EDTA, pH8.0) damping fluid room temperature;
4) secondary extracting:
The DNA IQ that 7uL suspends completely is added in the DNA solution extracted
tMdNA IQ in system
tMresin, high speed vortex oscillation 2 second, and carry out Magneto separate, described Magneto separate step comprises the steps:
1. pipe is placed on MagneSphere
on Magneto separate frame, Magneto separate carries out at once, all solution of careful removal, touches the resin on tube wall.Add 100 μ L lysis buffers, take off pipe from Magneto separate frame, and high speed vortex oscillation 3 second, pipe is put back on Magneto separate frame, and removes all lysates;
2. the 1x washings that 100 μ L prepare is added, pipe is taken off from Magneto separate frame, and high speed vortex oscillation 3 second, pipe is put back on Magneto separate frame, removes all washingss, uncap, pipe is placed on Magneto separate frame, air drying 8 minutes, adds 100 μ L elutriants, lid upper tube cap high speed vortex oscillation 3 second;
3. 65 DEG C of incubations 8 minutes.Take out the pipe of incubation, high speed vortex oscillation 3 second, be put into immediately on Magneto separate frame.
After separation terminates, DNA solution is transferred in selected container carefully.
Carry out pcr amplification and detect DNA, DNA is extracted in display.
Claims (1)
1. an extracting method of tanned animal sample hide DNA, is characterized in that: comprise the steps: successively
1) process of raw material:
1. by the zoological specimens hide through tanning process at normal temperatures with alcohol-pickled, more for subsequent use to no-sundries with distilled water flushing;
2. clip hide, uses Na
2hPO
4solution processes 20 ~ 28 hours under continuous oscillating condition, with distilled water flushing 2 ~ 3 times;
2) digest:
Soaked 1.5 ~ 2.5 hours by sample soak solution, abandon soak solution, shred with scissors as far as possible, be placed in centrifuge tube, add 500 μ l Digestive systems in centrifuge tube, 24 μ l 0.5mol/L EDTA, 6 μ l 20mg/ml Proteinase Ks, deposit 3-5 hour at 55 DEG C; Centrifugation, gets 200 μ l supernatant liquors in another centrifuge tube;
Described soak solution is 10mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L NaCl, pH8.0;
3) be separated, precipitate:
In centrifuge tube, add the saturated phenol of 200 μ lTris-, pH8.0, shake mixing 8 ~ 12 minutes lightly, centrifugation, repeats this step once; Moved to by supernatant in a new centrifuge tube, then add 200 μ l chloroforms, shake mixing 8 ~ 12 minutes lightly, centrifugation, gets supernatant and repeats this step once; Get supernatant to move in a new centrifuge tube, add the cold dehydrated alcohol of 2.5 times of volumes, shake a moment lightly, be placed in refrigerator under-20 DEG C of conditions freezing 25 ~ 35 minutes; Centrifugation, careful supernatant discarded; Draw 200 μ L70% washing with alcohol for a moment, centrifugation, careful supernatant discarded, opens centrifuge tube lid, makes ethanol evaporation 10-20 minute under room temperature; By dissolving DNA precipitation under 30 μ l TE damping fluid room temperatures, TE:10 mmol/L Tris-HCl, 10 mmol/L EDTA, pH8.0;
4) secondary extracting:
The DNA IQ that 7 μ L suspend completely is added in the DNA solution extracted
tMdNA IQ in system
tMresin, the high speed vortex oscillation several seconds, and carry out Magneto separate, be separated after terminating, DNA solution is transferred in container carefully;
5) carry out pcr amplification and detect DNA, DNA is extracted in display;
Magneto separate step in described secondary extracting comprises the steps:
1. pipe is placed on Magneto separate frame, Magneto separate carries out at once, the all solution of careful removal, do not touch the resin on tube wall, add 100 μ L lysis buffers, take off pipe from Magneto separate frame, and the high speed vortex oscillation several seconds, pipe is put back on Magneto separate frame, and removes all lysates;
2. add the 1x washings that 100 μ L prepare, take off pipe from Magneto separate frame, and the high speed vortex oscillation several seconds, pipe is put back on Magneto separate frame, removes all washingss; Uncap, is placed on Magneto separate frame, air drying by pipe, add 25-100 μ L elutriant, lid upper tube cap high speed vortex oscillation several seconds;
3. 65 DEG C of incubations 5 ~ 8 minutes, take out the pipe of incubation, the high speed vortex oscillation several seconds, are put on Magneto separate frame immediately, are separated and terminate;
Described step 2) in Digestive system be Promega company produce Nuclei Lysis Sol ' n reagent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310196655.6A CN103243089B (en) | 2013-05-24 | 2013-05-24 | Method for extracting deoxyribonucleic acid (DNA) from tanned animal specimen hide |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310196655.6A CN103243089B (en) | 2013-05-24 | 2013-05-24 | Method for extracting deoxyribonucleic acid (DNA) from tanned animal specimen hide |
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| CN103243089B true CN103243089B (en) | 2015-03-04 |
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| CN108410861B (en) * | 2018-05-16 | 2022-02-22 | 中国科学院古脊椎动物与古人类研究所 | Method and kit for extracting ancient DNA (deoxyribonucleic acid) in hide |
| CN111349628A (en) * | 2018-12-21 | 2020-06-30 | 苏州市产品质量监督检验院 | Membrane adsorption column method for extracting nucleic acid from natural leather and products thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270357A (en) * | 2008-03-11 | 2008-09-24 | 华东理工大学 | Method for extracting DNA from deep process type traditional Chinese medicine or traditional Chinese medicinal materials |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270357A (en) * | 2008-03-11 | 2008-09-24 | 华东理工大学 | Method for extracting DNA from deep process type traditional Chinese medicine or traditional Chinese medicinal materials |
Non-Patent Citations (2)
| Title |
|---|
| 冯慧,等.林麝熟制标本皮张DNA的提取及PCR分析.《陕西科技大学学报》.2011,第29卷(第3期),37-40. * |
| 贾学渊,等.鞣制皮张DNA的提取.《东北林业大学学报》.2007,第35卷(第11期),66-69. * |
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