CN111454940B - Method for efficiently extracting pig hair follicle DNA for high-throughput SNP typing - Google Patents
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Abstract
The invention discloses a method for efficiently extracting pig hair follicle DNA for high-throughput SNP typing, which is used for efficiently extracting complete genome DNA from pig hair follicles based on a novel phenol-chloroform method formed by improving a traditional phenol-chloroform method, and the obtained DNA has high purity and concentration and can be used for a high-throughput SNP typing technology. The method comprises the steps of collecting hair follicle tissues of pigs, adding tissue extract and proteinase K to digest the hair follicle tissues, and adding Tris saturated phenol, saturated phenol: chloroform: DNA was extracted with isoamyl alcohol (25:24:1) and chloroform, precipitated with glacial ethanol, washed with 70% ethanol, and air-dried to obtain a DNA sample. The genome DNA extracted by the method has good purity and integrity, completely meets the requirements of high-throughput SNP typing on the quality and the total amount of the DNA, has good economy and has wide application prospect.
Description
Technical Field
The invention relates to the field of molecular biology, and introduces a method for efficiently extracting pig hair follicle DNA for high-throughput SNP typing.
Background
The maturity of high-throughput SNP typing technology and the rapid development of molecular biology technology promote the continuous improvement of molecular breeding research level. Currently, molecular marker assisted breeding or whole genome selective breeding techniques are widely applied in pig breeding. The genetic variation information of the genome can be rapidly detected through high-throughput SNP chip typing, the whole genome selective breeding of the pig can be developed, genetic evolution analysis and whole genome association analysis can be developed, the important character candidate genes of the pig can be positioned, and the method has important application and scientific research values. And the extraction of high-quality complete genome DNA is the basis for high-throughput SNP typing and is the basic premise for further research on pig genetic breeding.
In the research of pig molecular genetic breeding, most researches adopt blood, ear tissues and the like as common materials for DNA extraction, but both sampling modes can generate great stress to pigs, and the pigs often bleed, so that the body of the pigs is damaged. Since the occurrence of African swine fever is first reported by Liaoning in China in 8 months in 2018, the method for collecting DNA of ear tissues and blood samples cannot be carried out in consideration of epidemic prevention safety. Compared with the method for collecting samples such as blood, tissues and the like, the pig hair follicle sample is easier to obtain, great damage and stress to individual pigs are avoided, and the risk of biosafety brought by sampling is greatly reduced. However, because the hair follicle tissue is less in quantity and high in protein content, the extracted genomic DNA is generally less in total quantity and low in quality, and the requirement of high-throughput SNP typing cannot be met. Therefore, the exploration of the extraction method for extracting high-quality and complete genome DNA from the pig hair follicle has important significance.
Phenol-chloroform extraction is a common method for extracting DNA in the laboratory, and can be used for epithelial, blood, muscle tissue, etc. The requirement of the high-throughput SNP typing technology on the quality of a DNA sample is that OD 260/280 is between 1.7 and 2.1, and the value of OD260/230 of a purer nucleic acid sample is more than 2.0. Methods for extracting DNA from pig hair follicles have been reported in the early days, for example, in 2010, Zhouyou English[1]The phenol-chloroform method is improved for extracting DNA from pig hair follicleThe point is that high salt and saturated phenol are used: chloroform: isoamyl alcohol (25:24:1) is used for extracting DNA, and tests show that the obtained DNA has low purity and poor repeatability. The next year, week construction, etc[2]The introduced method for rapidly extracting DNA from the pig hair follicle is characterized in that only saturated phenol is used: chloroform: the DNA was extracted with isoamyl alcohol (25:24:1), and the results showed that the mass of the obtained DNA sample was comparable to that of periphylon[1]The method of the people has no obvious improvement, and can not meet the requirement of high-throughput SNP typing technology on the quality of DNA samples.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for efficiently extracting pig hair follicle DNA for high-throughput SNP typing, the method is based on a novel phenol-chloroform method formed by improving the traditional phenol-chloroform method, can efficiently extract complete genome DNA from pig hair follicles, and the obtained DNA has high purity and concentration and can be used for high-throughput SNP typing.
In order to achieve the purpose, the technical scheme is as follows:
a method for efficiently extracting pig hair follicle DNA for high-throughput SNP typing is characterized by sequentially comprising the following steps: collecting hair follicles, digesting the hair follicles, extracting DNA, precipitating the DNA and washing; wherein, the extraction step of the DNA comprises the following steps of sequentially extracting Tris saturated phenol: chloroform: the DNA was extracted with a 25:24:1 mixture of isoamyl alcohol and chloroform.
The method of the invention preferably comprises the following steps in sequence:
(1) collecting hair follicles: 20 fresh pig hairs with intact hair follicles were harvested, and approximately 0.5cm long sections of the roots with hair follicles were cut with scissors and placed into 1.5mL centrifuge tubes.
(2) Digestion of hair follicles: adding tissue extract (50mmol/L Tris-Cl (pH8.0), 100mmol/L EDTA (pH8.0), 100mmol/L NaCl, 1% SDS) and proteinase K, fully mixing and centrifuging, digesting in a 56 ℃ water bath for 1-2 h, and flicking the sample with fingers 3-4 times to accelerate the digestion process;
(3) extraction of DNA: tris saturated phenol, saturated phenol: chloroform: extracting DNA with isoamyl alcohol (25:24:1) and chloroform;
(4) precipitation and washing of DNA: adding 2 times of supernatant volume of glacial ethanol to precipitate DNA as flocculent precipitate. Then, the DNA precipitate was washed with 70% ethanol, air-dried, and DNA was dissolved by adding an appropriate amount of TE (10mmol/L, pH8.0 Tris-Cl, 1mmol/L, pH8.0 EDTA), and stored at-20 ℃.
In step (2), 200. mu.L of the tissue extract was added, and 20. mu.L of proteinase K was added at a concentration of 20 mg/mL.
The specific process of DNA extraction in step (3): adding 180 mu L of deionized water into the centrifuge tube in the step (2), adding 400 mu L of Tris saturated phenol (pH is greater than 7.8) into the centrifuge tube, and uniformly mixing for 2-3 min by turning the centrifuge tube upside down by manual or automatic equipment, wherein the frequency is 60 times/min, so that the protein is fully denatured. Centrifuging at 12000g for 5min, and placing the supernatant in another new centrifuge tube; to the tube was added again 400 μ L of saturated phenol: chloroform: isoamyl alcohol (25:24:1), turning the centrifuge tube upside down, and mixing for 2-3 min to fully denature the protein. Centrifuge at 12000g for 5min, and take the supernatant in another new centrifuge tube. And finally adding 400 mu L of chloroform, inverting the centrifuge tube for 2-3 min, centrifuging 12000g for 5min, and taking the supernatant into another new centrifuge tube.
The specific process of DNA precipitation in step (4): adding 2 times of volume of glacial ethanol (pre-cooling in a refrigerator at-20 ℃ C.), and turning the centrifugal tube upside down for 2-3 min to separate out DNA as flocculent precipitate. 12000g centrifugal 4min, make DNA precipitation to the tube bottom of the centrifugal tube.
When washing with 70% glacial ethanol in the step (4), gently blowing up the DNA precipitate, centrifuging at 12000g for 4min, discarding the supernatant, repeating the steps once, sucking the residual ethanol in the centrifuge tube with a pipette, opening the centrifuge tube, placing the centrifuge tube in a fume hood for 10 min until the white precipitate in the tube becomes transparent particles, and adding a proper amount of TE to dissolve the DNA.
The tissue extract and proteinase K are added in the present invention in concentrations and amounts sufficient to allow sufficient hydrolysis of the hair follicle within one hour, releasing all the DNA. The deionized water is added into the digestive juice to ensure that the digestive juice and 400 mu L of phenol have equal volume reaction, thereby increasing the recovery volume of the supernatant containing DNA, increasing the yield of the DNA and reducing the DNA loss caused by two times of phenol extraction to the maximum extent. Meanwhile, chloroform is used after two times of extraction, and the step is to remove residual phenol in the DNA solution and effectively improve the purity of the DNA.
The total amount of DNA extracted by the method is sufficient, the purity is high, and impurities are few. Compared with the prior art, the method has the advantages that the OD260/230 value is greatly improved in the DNA purity measurement, the level of a pure nucleic acid sample can be reached, and the high-throughput SNP typing and other subsequent molecular biological tests can be completely met.
In the test process, the invention determines the reagent amount suitable for reaction, ensures the repeated effect of the test and the feasibility of batch extraction to a certain extent, avoids the waste of the reagent and has certain economical efficiency. In the test process, compared with the traditional phenol-chloroform method, the reaction and centrifugation time is greatly shortened, so that the whole reaction process can be carried out efficiently.
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FIG. 1 example 1 extraction of genomic DNA from porcine follicle tissue agarose gel electrophoresis detection notes: m is 2000bpMarker, and 1, 2, 4, 7 and 8 are 6 hair follicle samples; 2. 5, 6, 9, 10 are 12 hair follicle samples.
FIG. 2 example 4 extraction of genomic DNA from porcine follicle tissue agarose gel electrophoresis detection notes: m is 2000bpMarker, and A to H are 20 hair follicle samples.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention as claimed.
Example 1
The method is derived from Zhouqiong and the like, and utilizes a modified phenol-chloroform method to extract genome DNA from pig hair follicles, and comprises the following steps:
(1) 1-12 pig hairs with hair follicles are taken, the parts with the hair follicles at the roots, which are about 0.5cm, are cut by scissors and are put into a 0.5mL centrifuge tube.
(2) Adding tissue extract (50mmol/L Tris-Cl (pH8.0), 100mmol/L EDTA (pH8.0), 100mmol/L NaCl, 1% sodium dodecyl sulfate) 200. mu.L, proteinase K (20 mg/. mu.l) 5-8. mu.L, and mixing well. Digesting in water bath at 55 ℃ for 1-2 h, and flicking the sample with fingers in the middle to accelerate the digestion process.
(3) Then, 60. mu.L of 5mol/L NaCl, saturated phenol: chloroform: isoamyl alcohol (25:24:1) 260. mu.L, and the mixture is turned upside down and mixed in a centrifuge tube for 5min to fully denature the protein. Centrifuging at 10000r/min for 5min, and taking the supernatant in another new centrifuge tube.
(4) Adding 2 times of supernatant volume of glacial ethanol, and slightly inverting the centrifuge tube to precipitate the DNA as flocculent precipitate. Centrifuging at 10000r/min for 5min to precipitate the DNA to the bottom of the centrifuge tube. Then, the DNA pellet was washed with 70% ethanol 2 times, air-dried, and 40. mu.L of TE (10mmol/L of Tris-Cl, pH8.0, 1mmol/L of EDTA, pH8.0) was added to dissolve the DNA, and the DNA was stored at-20 ℃.
TABLE 1 example 1 extraction of genomic DNA concentration, Total amount and purity from porcine follicle tissue
Example 2
The method is derived from the improved experiment of example 1, and based on the results in Table 1, we guess that high salt, although it can play a role in rapidly precipitating proteins, will also have a certain effect on the purity of DNA samples, making the ratio of OD260/230 lower. Therefore, 60. mu.L of NaCl was added at different gradient concentrations for step (3) in example 1 and a control group was established.
Group A as control group, adding deionized water 60 μ L, and saturated phenol/chloroform/isoamyl alcohol (25:24:1)260 μ L;
group B was supplemented with 1mol/L NaCl 60. mu.L, saturated phenol: chloroform: isoamyl alcohol (25:24:1) 260. mu.L;
group C, 2mol/L NaCl 60. mu.L, saturated phenol: chloroform: isoamyl alcohol (25:24:1) 260. mu.L;
group D, 3mol/L NaCl 60. mu.L, saturated phenol: chloroform: isoamyl alcohol (25:24:1) 260. mu.L;
group E was supplemented with 4mol/L NaCl 60. mu.L, saturated phenol: chloroform: isoamyl alcohol (25:24:1) 260. mu.L;
group F was supplemented with 5mol/L NaCl 60. mu.L, saturated phenol: chloroform: isoamyl alcohol (25:24:1) 260. mu.L.
The subsequent reaction was carried out, and the rest of the procedure was the same as in example 1.
TABLE 2 example 2 extraction of genomic DNA concentration, Total amount and purity from porcine follicle tissue
Example 3
The method results from an improved experiment on example 2, in example 2, we found that the ratio of OD260/230 of the group A of the control group is the highest, but the requirement of high-throughput SNP typing on the purity of the DNA sample cannot be met. In order to increase the OD260/230 ratio, we continued the optimization based on step (3) A set in example 2, using Tris saturated phenol, saturated phenol: chloroform: isoamyl alcohol (25:24:1) and chloroform were extracted three times in this order, and the reaction amount of the reagent was determined. The method comprises the following steps:
(1) taking 12 fresh pig hairs with complete hair follicles, shearing the parts with hair follicles at the roots and the length of about 0.5cm by using scissors, and putting the parts into a 1.5mL centrifuge tube;
(2) adding 200 mu L of tissue extract (50mmol/L Tris-Cl (pH8.0), 100mmol/L EDTA (pH8.0), 100mmol/L NaCl, 1% sodium dodecyl sulfate) and 20 mu L of protease K (20mg/mL), fully mixing and centrifuging, digesting in a water bath at 55 ℃ for 1-2 h, and flicking the sample with fingers for 3-4 times to accelerate the digestion process;
(3) adding 180 mu l of deionized water into the centrifuge tube, adding 400 mu l of Tris saturated phenol (pH is more than 7.8) into the centrifuge tube, and uniformly mixing for 2-3 min by turning the centrifuge tube upside down by manual or automatic equipment, wherein the frequency is 60 times/min. Centrifuging at 12000g for 5min, and placing the supernatant in another new centrifuge tube; to the tube was added again 400. mu.l of saturated phenol: chloroform: isoamyl alcohol (25:24:1), turning the centrifuge tube upside down, and mixing for 2-3 min to fully denature the protein. Centrifuge at 12000g for 5min, and take the supernatant in another new centrifuge tube. And finally adding 400 mu l of chloroform, inverting the centrifuge tube upside down for 2-3 min, centrifuging 12000g for 5min, and taking the supernatant into another new centrifuge tube.
(4) Adding 2 times of supernatant volume of glacial ethanol (pre-cooling in a refrigerator at-20 ℃ C.), and inverting the centrifuge tube for 2-3 min to precipitate DNA as flocculent precipitate. Centrifuging at 12000g for 4min to precipitate the DNA to the bottom of the centrifuge tube;
(5) the DNA pellet was washed 2 times with 70% glacial ethanol, centrifuged 4min at 12000g each time and then air dried naturally, and 40. mu.L of TE (10mmol/L of Tris-Cl, pH8.0, 1mmol/L of EDTA, pH8.0) was added to dissolve the DNA and stored at-20 ℃.
TABLE 3 example 3 extraction of genomic DNA concentration, Total amount and purity from 12 porcine hair follicle tissues
Example 4
The test results of example 3 are shown in Table 3, in which the DNA samples obtained by the method were of good purity, but the total amount of DNA obtained was reduced. Therefore, in order to increase the total amount of DNA obtained from a single sample, the present case only adjusted the number of hair follicles in step (1) based on example 3.
The specific steps are changed as follows:
(1) taking 20 fresh pig hairs with complete hair follicles, shearing the parts with hair follicles at the roots and the length of about 0.5cm by using scissors, and putting the parts into a 1.5mL centrifuge tube; the rest of the procedure was the same as in example 1.
As can be seen from Table 1, the OD260/230 ratio in example 1 was low, indicating that the DNA sample extracted by this method had many impurities. And it can be seen from FIG. 1 that the electrophoresis strip of the DNA sample is blurred, which may be the result of the DNA sample containing too high salt. By way of example 2, we added NaCl at different gradient concentrations to step (3) of example 1 and established a control group, and the results are shown in Table 2, in 7 groups, the ratio of OD260/230 was the highest in group A of DNA samples in the control group without NaCl, while the ratio of OD260/230 tended to decrease in the remaining 6 groups as the concentration of NaCl was increased. Subsequently, based on the embodiments 1 and 2, the procedure was optimized to make the ratio of OD260/230 of the obtained DNA sample above 2.0, which indicates that the DNA extracted from the pig hair follicle using the embodiment 3 has better purity, but the total amount of DNA is reduced due to the increase of the extraction times. Therefore, in example 4, the number of hair follicle samples is increased from 12 to 20, and the test results are shown in table 4, so that the obtained DNA samples have good purity, concentration and total amount, meet the requirements of high-throughput SNP typing, and ensure the feasibility of batch extraction.
TABLE 4 example 4 extraction of genomic DNA concentration, Total amount and purity from 20 porcine follicle tissues
Reference:
[1] wang Shen Ying, Shu Ying, Feng Li Xia, Wang Huai, Zhang Qin, extracting genomic DNA [ J ] from pig hair follicle by using a modified phenol-chloroform method, inheritance, 2010, 32(07): 752-.
[2] Zhou construction, Chen Xin, Li Ming, Xie Fu, Liu directional wave, effective method for quickly extracting genome DNA from pig hair follicle [ J ]. northwest agro-academy 2011, 20(08):12-15.
Claims (7)
1. A method for efficiently extracting pig hair follicle DNA for high-throughput SNP typing is characterized by sequentially comprising the following steps: collecting hair follicles, digesting the hair follicles, extracting DNA, precipitating the DNA and washing; wherein, the extraction step of the DNA comprises the following steps of sequentially extracting Tris saturated phenol: chloroform: isoamyl alcohol =25: 24:1 mixture and chloroform were used for three extractions of DNA.
2. The method according to claim 1, characterized by comprising the following steps in sequence:
(1) collecting hair follicles: collecting 18-25 fresh pig hairs with complete hair follicles, shearing a 0.5-0.7 cm long part with the hair follicles at the root, and putting the part into a 1.5mL centrifuge tube;
(2) digestion of hair follicles: adding the tissue extract and protease K, fully mixing uniformly and centrifuging, digesting in a water bath at 56 ℃ for 1-2 h, and flicking the sample with fingers 3-4 times in the middle to accelerate the digestion process; the tissue extract consists of 50mmol/L Tris-Cl with pH of 8.0, 100mmol/L EDTA with pH of 8.0, 100mmol/L NaCl and 1 percent SDS;
(3) extraction of DNA: respectively sequentially adding Tris saturated phenol, saturated phenol: chloroform: extracting DNA for three times by using a mixed solution of isoamyl alcohol =25: 24:1 and chloroform;
(4) precipitation and washing of DNA: adding glacial ethanol to precipitate DNA into flocculent precipitate, washing the DNA precipitate with 70% ethanol, air drying, adding appropriate amount of TE to dissolve DNA, and storing at-20 deg.C; the TE composition is 10mmol/L Tris-Cl with pH8.0 and 1mmol/L EDTA with pH 8.0.
3. The method of claim 2, wherein: collecting hair follicle samples in the step (1), drawing hairs along the growth direction of the hairs, checking whether hair follicles exist at roots, counting 20 hairs, collecting the samples, and storing at-20 ℃.
4. The method of claim 2, wherein: the amount of the tissue extract added in the step (2) was 200. mu.L, and 20. mu.L of proteinase K was added at a concentration of 20 mg/mL.
5. The method of claim 2, wherein: the specific process of DNA extraction in step (3): adding 180 mu L of deionized water into the centrifugal tube in the step (2), adding 400 mu L of Tris saturated phenol with the pH value of more than 7.8 into the centrifugal tube, turning the centrifugal tube upside down, uniformly mixing for 2-3 min to fully denature the protein, centrifuging for 5min at 12000g, and taking the supernatant into another new centrifugal tube; to the tube was added again 400 μ L of saturated phenol: chloroform: the mixed solution of isoamylol =25: 24:1 is mixed for 2-3 min by turning the centrifuge tube upside down to fully denature the protein, 12000g is centrifuged for 5min, and the supernatant is taken out to be put into another new centrifuge tube; and finally adding 400 mu L of chloroform, inverting the centrifuge tube for 2-3 min, centrifuging 12000g for 5min, and taking the supernatant into another new centrifuge tube.
6. The method of claim 2, wherein: the specific process of DNA precipitation in step (4): adding 2 times of volume of glacial ethanol, inverting the centrifuge tube for 2-3 min to precipitate DNA into flocculent precipitate, and centrifuging 12000g for 4min to precipitate DNA to the bottom of the centrifuge tube.
7. The method of claim 2, wherein: the specific process of the step (4): when the DNA is washed by 70% glacial ethanol, the DNA precipitate is blown up gently, 12000g of DNA precipitate is centrifuged for 4min, and the supernatant is discarded; repeating the steps once, sucking the residual ethanol in the centrifugal tube by using a pipette, opening the centrifugal tube, placing the centrifugal tube under a fume hood for 10 minutes until white precipitates in the centrifugal tube become transparent particles, adding a proper amount of TE (potassium titanate) to dissolve DNA, and storing at-20 ℃.
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| CN105950614A (en) * | 2016-07-21 | 2016-09-21 | 中南大学 | Method for extracting hair DNA |
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