A method for efficiently extracting pig hair DNA

Abstract

The present invention relates to a method for efficiently extracting porcine hair DNA, comprising the following steps: hair follicle collection, hair follicle digestion, DNA extraction, precipitation and washing. The DNA extraction is carried out twice. The first extraction is with Tris-saturated phenol, and the second extraction is with a mixture of saturated phenol: chloroform: isoamyl alcohol = 25:24:1. Then, isopropanol and 10 M ammonium acetate are added to the secondary extraction solution for the first precipitation of DNA, and ice ethanol is added to the precipitate for the second precipitation of DNA, followed by subsequent washing and other treatments. Using the method of the present invention, porcine hair DNA of Duroc, Large White pigs and Landrace pigs can be quickly and effectively extracted, and the extraction quality meets the requirements of DNA for gene chip sequencing.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a method for efficiently extracting porcine hair DNA. Background Art

[0002] With the development of SNP chips and sequencing technologies, genomic selection methods have developed rapidly since their proposal in 2001 and have become an application hotspot in the field of animal breeding, bringing revolutionary changes to animal breeding. In the field of pig breeding, genomic selection can improve the accuracy of selection, accelerate genetic progress, benefit from early evaluation of candidate individuals, and early elimination, and is attractive for traits that are difficult to improve by conventional breeding. The prerequisite for genomic selection is to obtain the genomic information of pigs. Pig breeding industry practitioners usually collect ear and blood samples of pigs to extract total DNA. The collection of ear tissue requires clamping the ear part with forceps and cutting off a small piece of ear tissue with scissors. The collection of blood requires pulling the pig's mouth with a pig tether and collecting blood from the pig's neck. Inexperienced blood collectors often need to puncture multiple times to collect, which is time-consuming, laborious, and difficult. Whether collecting ear tissue or blood, it will cause certain stress to pigs, and in some cases, it will even lead to non-cooperation in the breeding farm, delaying the progress of breeding work. Hair samples are an easily obtainable sample, which is easy to collect and does not cause damage or stress to pigs. At the same time, the storage conditions are simple, and only a dry sealed bag is needed for long-term storage. However, due to the low DNA content and high keratin content in pig hair, which is not easily digested, the genomic DNA obtained from pig hair usually has a small total amount and low quality, and cannot meet the requirements of gene chip sequencing. Therefore, a highly universal method for obtaining high-quality genomic DNA extraction from pig hair can accelerate the progress of breeding work and has important significance.

[0003] The phenol-chloroform extraction method is a classic and commonly used method for extracting DNA in laboratories, applicable to various tissues, including epithelium, blood, muscle, etc. Gene chip sequencing and whole genome sequencing have high requirements for sample DNA. The total amount of DNA needs to be greater than 2.0 μg, and the purity OD 260 / 230 should be greater than 1.95. Methods for extracting genomic DNA from pig hair have been reported. Among them, in Chinese Patent 202010281947.X, a method for efficiently extracting porcine follicle DNA for high-throughput SNP genotyping performs three extractions. Its feature is adding deionized water in the first phenol extraction to increase DNA recovery and reduce DNA loss. The extracted DNA has high purity and reaches the level of pure nucleic acid samples, and can be used for high-throughput SNP genotyping. However, when this method is applied to three common pig breeds, Duroc, Yorkshire, and Landrace, with short follicle diameters and short follicle lengths, the total amount of extracted DNA is often low, and the repeatability of purity determination is poor, unable to meet the requirements of gene chip sequencing for DNA. Summary of the Invention

[0004] To solve the above problems, the present invention proposes a method for efficiently extracting porcine hair DNA, which is an improvement on the existing porcine hair DNA extraction method to improve the extraction efficiency of porcine hair DNA. Using this method, the porcine hair DNA of Duroc, Yorkshire, and Landrace pigs can be quickly and effectively extracted, and the extraction quality meets the requirements of DNA for gene chip sequencing.

[0005] The method for efficiently extracting porcine hair DNA of the present invention includes the following steps: hair follicle collection, hair follicle digestion, DNA extraction, precipitation, and washing. The DNA extraction is performed twice. The first extraction is with Tris-saturated phenol, and the second extraction is with a mixture of saturated phenol:chloroform:isoamyl alcohol = 25:24:1. Then, isopropanol and 10 M ammonium acetate are added to the secondary extraction solution to perform the first precipitation of DNA, and ice ethanol is added to the precipitate to perform the second precipitation of DNA, and then subsequent washing and other treatments are carried out.

[0006] Furthermore, when the number of hair follicles is 20, the usage amounts of isopropanol and 10 M ammonium acetate are 360 μL of isopropanol and 40 μL of 10 M ammonium acetate.

[0007] Furthermore, the porcine hair comes from Duroc, Yorkshire, and Landrace pigs.

[0008] Furthermore, the method for efficiently extracting porcine hair DNA of the present invention includes the following steps:

[0009] S1. Hair follicle collection: Take 20 porcine hairs with intact hair follicles, cut about 0.5 cm of the part with hair follicles at the root with scissors and put it into a 1.5 mL centrifuge tube.

[0010] S2. Hair follicle digestion: Add 380 μL of tissue extraction solution and 20 μL of proteinase K to the above centrifuge tube, mix well and centrifuge to completely immerse the hair follicles. Digest in a water bath at 56 °C for 1 - 2 hours, and gently flick the sample with your finger during the process to accelerate digestion.

[0011] S3. DNA extraction: First, add 400 μL of Tris-saturated phenol to the centrifuge tube obtained in S2, invert and mix well for 3 min, and centrifuge at 12,000 g for 5 min at 4 °C. Then, take the supernatant and transfer it to another new 1.5 mL centrifuge tube, add 400 μL of a mixture of Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1 to it, invert and mix well for 3 min, and centrifuge at 12,000 g for 5 min at 4 °C. Take the supernatant and transfer it to another new 1.5 mL centrifuge tube.

[0012] S4. Precipitate DNA: Add 360 μL of isopropanol and 40 μL of 10 M ammonium acetate to the centrifuge tube obtained in S3, invert the tube up and down to mix well for 3 min, centrifuge at 12,000 g for 5 min at 4 °C to precipitate the DNA to the bottom of the centrifuge tube, and pour off the supernatant; then add ice-cold ethanol pre-cooled to -20 °C with a volume twice that of the supernatant to the centrifuge tube from which the supernatant has been poured off, invert the centrifuge tube 2 - 3 min, centrifuge at 12,000 g / min for 4 min, and discard the supernatant.

[0013] S5. Wash: Wash the DNA precipitate in the centrifuge tube obtained in S4 twice with 70% ethanol, centrifuge at 12,000 g for 4 min each time, suck out the residual ethanol in the centrifuge tube with a pipette, open the centrifuge tube and place it under the fume hood for 10 minutes until the white precipitate in the tube becomes transparent particles, dissolve the DNA in an appropriate amount of TE buffer, pipette to mix well, and then store it in a -20 °C refrigerator for a long time.

[0014] Furthermore, the tissue extraction solution is composed of 50 mmol / L Tris-CL with pH 8.0, 100 mmol / L EDTA with pH 8.0, 100 mmol / L NaCl, and 1% SDS.

[0015] Furthermore, the pig hair comes from the back of the pig. When collecting, follow the direction of the hair and retain the hair with hair follicles, and store it dry at room temperature in a sealed bag.

[0016] The beneficial effects of the present invention are as follows:

[0017] 1. The present invention changes the three-time extraction and one-time precipitation of DNA to two-time extraction and two-time precipitation. It does not use chloroform alone for extraction. For the first precipitation, isopropanol and ammonium acetate are used, and then ice-cold ethanol is used for secondary precipitation, which can significantly increase the total amount of DNA extracted. Especially for pig hair of pig breeds with less DNA content, the quality and efficiency of DNA extraction are greatly improved.

[0018] 2. The present invention discovers for the first time that using a combination of isopropanol and ammonium acetate to replace chloroform after the second extraction can well remove the residual phenol and improve the purity of DNA, while increasing the total amount of DNA extracted, and gives the best combination ratio of isopropanol and ammonium acetate.

[0019] 3. To ensure the DNA extraction efficiency, in the industry, 20 pig hairs are used as a sample for pig hair DNA extraction. When extracting the DNA of 20 pig hairs of the mainstream pig breeds in pig farms, namely Large White, Landrace, and Duroc, using the existing technology, the total amount of pig hair DNA extracted is low, the purity is low, and there are many impurities, which does not meet the requirements of gene chip sequencing. However, when this invention is applied to the hairs of Duroc, Landrace, and Large White pigs, the total amount of genomic DNA extracted is far greater than 2.0 μg, with high purity and few impurities. Compared with the existing technology, the total amount of DNA extracted by the method of this invention is significantly increased, and OD 260 / 230 is stably maintained at a level greater than 1.95. The extracted DNA not only meets the requirements of routine PCR experiments in the laboratory but also meets the high requirements of gene chip sequencing and whole genome sequencing.

[0020] 4. During the experiment of this invention, Large White, Landrace, and Duroc, which are common in the pig breeding industry, are selected for hair DNA extraction, and DNA is successfully extracted from all of them, ensuring the universality of this method. At the same time, in actual breeding, in cooperation with a pig farm in Henan, the pig farm staff sampled, and the experimental staff extracted DNA. In this mode, a total of 1147 DNA samples were extracted, and 1127 of them met the requirements of subsequent chip sequencing and re-sequencing, with a qualified rate as high as 98.27%, indicating that this method has good stability. Description of the Drawings

[0021] Figure 1 Gel electrophoresis diagram of the pig hair DNA of 9 samples extracted in Example 3. Detailed Embodiments

[0022] The present invention will be further described below in conjunction with embodiments.

[0023] Example 1:

[0024] Using the method for efficient extraction of pig hair follicle DNA for high-throughput SNP genotyping in Chinese Patent No. 202010281947.X to extract the pig hair DNA of Duroc, Large White, and Landrace pigs, the steps are as follows:

[0025] (1) Take 20 fresh pig hairs with complete hair follicles from three Duroc, Large White, and Landrace pigs each, and use scissors to cut about 0.5 cm of the part with hair follicles at the root and put it into a 1.5 mL centrifuge tube;

[0026] (2) Add 200 μL of tissue extraction solution (50 mmol / L Tris-CL (pH 8.0), 100 mmol / L EDTA (pH 8.0), 100 mmol / L NaCl, 1% SDS) and 20 μL of proteinase K (20 mg / μL), mix well and centrifuge, and digest in a water bath at 56 °C for 1 - 2 hours, and gently flick the sample with your finger during the process to accelerate the digestion process;

[0027] (3) Add 180 μL of deionized water and 400 μL of Tris-saturated phenol (pH>7.8) to the tube, and mix by inverting the tube for 2-3 minutes at a frequency of 60 times / minute. Centrifuge at 12,000 g for 5 minutes, and take the supernatant into another new centrifuge tube; add 400 μL of Tris-saturated phenol: chloroform: isoamyl alcohol (25:24:1) to the tube, and mix by inverting the tube for 5 minutes to fully denature the protein. Centrifuge at 12,000 g / min for 5 minutes, and take the supernatant into another new centrifuge tube; finally, add 400 μL of chloroform, and mix by inverting the tube for 3 minutes to allow the DNA to precipitate in the form of flocs. Centrifuge at 12,000 g for 5 minutes at 4°C to allow the DNA to precipitate to the bottom of the centrifuge tube, and discard the supernatant;

[0028] (4) Add 2 times the volume of supernatant, pre-cooled ice ethanol at -20℃, turn the centrifuge tube upside down for 2-3 minutes, and centrifuge at 12000g / min for 4 minutes. Then rinse the DNA precipitate twice with 70% ethanol, centrifuging at 12000g for 4 minutes each time. Use a pipette to absorb the remaining ethanol in the centrifuge tube, open the centrifuge tube and place it in a fume hood for 10 minutes until the white precipitate in the tube turns into transparent particles, add an appropriate amount of TE buffer to dissolve the DNA, blow and mix with the pipette tip, and store it in a -20℃ refrigerator for long-term storage if necessary.

[0029] (5) Instrument calibration before DNA detection: Use a pipette to draw 1.5 μL of TE buffer onto the sample loading area of ​​the Nanodrop 2000 instrument for blank calibration. When the nucleic acid concentration value is between -0.1 and 0.1, stop calibration and start detection.

[0030] (6) DNA detection: Use a pipette to draw 1.5 μL of DNA solution onto the sample loading area of ​​the Nanodrop 2000 instrument, and detect the concentration and absorbance of the DNA.

[0031] Table 1 DNA concentration, total amount and purity extracted from hair tissues of Large White pigs, Landrace pigs and Duroc pigs using existing methods

[0032]

[0033] According to the results in Table 1, the total amount of DNA extracted from 20 hairs of Large White pigs, Landrace pigs and Duroc pigs using the existing technology is low, most of which are less than 2.0 μg, and most of the OD260 / 230 are less than 1.95, with poor purity, which cannot meet the requirements of gene chip sequencing for DNA.

[0034] Embodiment 2:

[0035] This method originated from the improved experiment on 202010281947.X. The pigs used in the method described in 202010281947.X may be local breed pigs. Compared with Yorkshire pigs, Landrace pigs, and Duroc pigs, local breed pigs have larger hair follicle diameters and hair diameters, and their hair follicle tissues are relatively complete. The total amount of DNA in pig hair is more, so better extraction results are obtained. However, the hair follicle tissues of common commercial breed pigs such as Yorkshire pigs, Landrace pigs, and Duroc pigs are smaller. As can be seen from Example 1, the DNA extraction effect of this method on Yorkshire pigs, Landrace pigs, and Duroc pigs is poor. It may be that the multiple extractions of this method cause more DNA loss and are not suitable for hair with less DNA content itself. At the same time, when adding chloroform for extraction, the upper and lower layer interfaces of the solution are not obvious, and it is easy to suck the lower part during extraction, thus introducing more impurities and resulting in poor stability of the extracted DNA purity.

[0036] In view of the loss of the total amount of DNA caused by multiple extractions, the means of precipitating DNA is used to replace the third extraction using chloroform. In DNA precipitation experiments, pre-cooled absolute ethanol, pre-cooled isopropanol, and NaCl solution with a final concentration of 0.1 - 0.25 mol / L are usually used. In this example, the following equal-volume reagents are used to replace the chloroform added in the last step of Example 1(3), and the remaining steps are the same as those in Example 1.

[0037] Group A adds 400 μL of pre-cooled absolute ethanol

[0038] Group B adds 400 μL of pre-cooled isopropanol

[0039] Group C adds 400 μL of 0.1 mol / L NaCl solution

[0040] Group D adds 400 μL of 0.25 mol / L NaCl solution

[0041] Group E adds 400 μL of 0.5 mol / L NaCl solution

[0042] Group F adds 400 μL of 1 mol / L NaCl solution

[0043] And for one hair sample with relatively poor effect among the three breeds (Yorkshire pig No. 3, Landrace pig No. 1, Duroc pig No. 3), DNA extraction is carried out.

[0044] Table 2 DNA concentration, total amount, and purity extracted from the hair tissue of Yorkshire pig No. 3

[0045]

[0046] Table 3 DNA concentration, total amount, and purity extracted from the hair tissue of Landrace pig No. 1

[0047]

[0048] Table 4 DNA Concentration, Total Amount and Purity Extracted from the Hair Tissue of Duroc No. 3 Pig

[0049]

[0050] In Example 2, we found that reducing the number of extractions often increases the total amount of DNA obtained. Comparatively, the use of pre-cooled isopropanol is superior to pre-cooled ethanol, with a higher total amount and purity of the extracted DNA. Compared with the addition of NaCl solution, the total amount of DNA extracted in the pre-cooled isopropanol group is slightly lower, but the purity is much higher than that of groups C, D, E, and F. Overall, isopropanol has the best precipitation effect on porcine hair DNA.

[0051] Example 3:

[0052] This method is derived from the improvement experiment of Example 2. By using the method of adding isopropanol to precipitate DNA obtained in Example 2 to replace the third extraction with chloroform, the effect is better than that of other precipitants. However, simply adding isopropanol still cannot meet the requirements of gene chip sequencing and whole genome sequencing. Therefore, the B group in step (3) of Example 2 was optimized.

[0053] Considering that although isopropanol can precipitate DNA and remove impurities in the second extraction, the addition of salt may better play its role. As an ammonium salt, ammonium acetate can combine with SDS in the solution through the ammonium ions it carries to form a slightly soluble substance. This process helps to reduce the charge on the surface of DNA and promote the precipitation of DNA. It can also increase the precipitation efficiency of DNA by neutralizing the charge on the surface of DNA, thereby improving the purity and yield of DNA. At the same time, ammonium acetate can also provide acetate ions, which helps to maintain the pH stability during the extraction process and the stability of DNA molecules. Therefore, an appropriate amount of ammonium acetate was added to isopropanol to precipitate DNA. To determine the best combination of isopropanol and ammonium acetate, different concentration gradients were set, and DNA was extracted from the three samples described in Example 2, with the remaining steps remaining unchanged. The combinations of isopropanol and ammonium acetate used are as follows:

[0054] Group A added 380 μL of isopropanol and 20 μL of 10 M ammonium acetate;

[0055] Group B added 370 μL of isopropanol and 30 μL of 10 M ammonium acetate;

[0056] Group C added 360 μL of isopropanol and 40 μL of 10 M ammonium acetate;

[0057] Group D added 350 μL of isopropanol and 50 μL of 10 M ammonium acetate;

[0058] Group E added 340 μL of isopropanol and 60 μL of 10 M ammonium acetate;

[0059] Table 5 DNA concentration, total amount and purity in pig hair tissues of Large White Pig No. 3 extracted using different combinations of isopropanol and ammonium acetate

[0060]

[0061] Table 6 DNA concentration, total amount and purity in pig hair tissues of Landrace Pig No. 1 extracted using different combinations of isopropanol and ammonium acetate

[0062]

[0063] Table 7 DNA concentration, total amount and purity in pig hair tissues of Duroc Pig No. 3 extracted using different combinations of isopropanol and ammonium acetate

[0064]

[0065] As can be seen from Tables 5, 6 and 7, adding ammonium acetate to isopropanol significantly increased the total amount of DNA obtained. However, ammonium acetate is essentially a salt, and adding an excessive amount will cause the extracted DNA to contain too much salt, thereby reducing the extraction purity. Considering the comprehensive extraction effect, Group C, which uses 360 μL of isopropanol and 40 μL of 10 M ammonium acetate to extract DNA, has the best effect, meeting the requirements for the total amount and purity in gene chip sequencing and whole genome sequencing.

[0066] Example 4:

[0067] To verify the universality of this method, DNA was extracted from the remaining 6 samples. Except that 400 μL of chloroform used in the third extraction of Example 1 was replaced with 360 μL of isopropanol and 40 μL of 10 M ammonium acetate, the remaining steps were the same as those in Example 1. The results of all samples extracted by this method were summarized, and the results are shown in Table 5.

[0068] Table 5 DNA concentration, total amount and purity in pig hair tissues of different samples extracted using the method of the present invention

[0069]

[0070] As can be seen from Table 5, for the extraction of pig hair DNA of the three breeds of Large White Pig, Landrace Pig and Duroc Pig, the purity OD260 / 230 can all be above 1.95, indicating high purity and few impurities of the extracted DNA. At the same time, the total amount of extracted DNA has increased, and all are greater than 2.0 μg. In addition, a 1% concentration agarose gel was prepared to detect the integrity of the DNA of the 9 samples in this Example 4. As Figure 1 shown in the gel electrophoresis diagram, the DNA bands of the pig hair of the 9 samples of the three breeds extracted by the present invention are clear, without tailing phenomenon, with good integrity and no degradation, meeting the requirements of gene chip sequencing and whole genome sequencing.

[0071] The method of the present invention takes into account that multiple extractions will reduce the total amount of DNA extracted and increase the contamination of impurities. At the same time, the DNA content in the hair of three pig breeds, namely Large White pigs, Landrace pigs and Duroc pigs, is relatively low. Therefore, the number of DNA extractions is reduced and the step of precipitating DNA with isopropanol and ammonium acetate is added, which increases the amount of DNA precipitated, making the extracted DNA meet the requirements of gene chip sequencing and whole genome sequencing, and improving the extraction efficiency while reducing the experimental time.

[0072] Example 5: Apply the method of the present invention to practice:

[0073] For 1147 pig hair samples provided by a certain pig farm in Henan in cooperation, including 822 Large White pigs, 248 Landrace pigs and 77 Duroc pigs, DNA extraction is carried out, including the following steps:

[0074] S1. Follicle collection: Take 20 pig hairs with intact follicles, cut about 0.5 cm of the part with follicles at the root with scissors and put it into a 1.5 mL centrifuge tube;

[0075] S2. Follicle digestion: Add 380 μL of tissue extraction solution and 20 μL of proteinase K to the above centrifuge tube, mix well and centrifuge to completely immerse the follicles. Digest in a water bath at 56 °C for 2 hours, and gently flick the sample with your finger during the process to accelerate digestion;

[0076] S3. DNA extraction: First, add 400 μL of Tris-saturated phenol to the centrifuge tube obtained in S2, invert and mix well for 3 min, and centrifuge at 12,000 g for 5 min at 4 °C; then take the supernatant and transfer it to another new 1.5 mL centrifuge tube, add 400 μL of a mixed solution of Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1 to it, invert and mix well for 3 min, and centrifuge at 12,000 g for 5 min at 4 °C; take the supernatant and transfer it to another new 1.5 mL centrifuge tube;

[0077] S4. DNA precipitation: Add 360 μL of isopropanol and 40 μL of 10 M ammonium acetate to the centrifuge tube obtained in S3, invert and mix well for 3 min, and centrifuge at 12,000 g for 5 min at 4 °C to precipitate the DNA to the bottom of the centrifuge tube, and pour off the supernatant; then add ice-cold ethanol pre-cooled at -20 °C with a volume twice that of the supernatant to the centrifuge tube from which the supernatant has been poured off, invert the centrifuge tube for 2 - 3 min, and centrifuge at 12,000 g / min for 4 min, and discard the supernatant;

[0078] S5, Washing: Rinse the DNA precipitate in the centrifuge tube obtained in S4 twice with 70% ethanol, centrifuge at 12,000 g for 4 min each time, aspirate the residual ethanol in the centrifuge tube with a pipette, open the centrifuge tube and place it under the fume hood for 10 minutes until the white precipitate in the tube becomes transparent particles, dissolve the DNA in an appropriate amount of TE buffer, pipette to mix evenly, and then store it in a -20°C refrigerator for long-term preservation.

[0079] Send the above-extracted 1,147 DNA samples to Shijiazhuang BoruiDi Company for whole-genome sequencing. The sequencing results show that among them, there are 812 Large White pig samples, 241 Landrace pig samples, and 74 Duroc pig samples with Q30 ≥ 80% and output ≥ 90%. Q30 represents the probability of base recognition error being 0.1%, that is, at least 80% of the sequencing base quality values are greater than 99.9%; Q30 ≥ 80% and output ≥ 90% indicate that the accuracy of the base sequence measured from this sample is extremely high. Therefore, it shows that a total of 1,127 DNA samples extracted from the pig hairs of Large White pigs, Landrace pigs, and Duroc pigs using the method of the present invention meet the sequencing requirements, are successfully sequenced, and the qualified rate is as high as 98.27%.

[0080] In summary, on the basis of the existing technology, the method of the present invention, after improvement, can significantly improve the purity, concentration, and total amount of DNA samples extracted from the pig hairs of three pig breeds with relatively low DNA content in the hair follicles of Large White pigs, Landrace pigs, and Duroc pigs. The extracted DNA all meets the requirements of gene chip sequencing and whole-genome sequencing, with good repeatability and reliable results.

[0081] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0082] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present invention.

[0083] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for efficiently extracting pig hair DNA, comprising the following steps: Hair follicle collection, hair follicle digestion, DNA extraction, precipitation and washing, characterized in that the DNA is extracted twice, the first time is extracted with Tris saturated phenol, and the second time is extracted with a mixed solution of saturated phenol: chloroform: isoamyl alcohol = 25: 24: 1; then isopropanol and 10M ammonium acetate are added to the secondary extract to precipitate the DNA for the first time, and ice ethanol is added to the precipitate to precipitate the DNA for the second time, followed by subsequent washing treatment; when the number of hair follicles is 20, the amount of isopropanol and 10M ammonium acetate used is 360μL isopropanol and 40μL 10M ammonium acetate; the pig hair comes from Duroc, Large White and Landrace pigs.

2. The method for efficiently extracting pig hair DNA according to claim 1, characterized in that: The following steps are involved: S1. Hair follicle collection: Take 20 pig hairs with complete hair follicles, cut 0.5 cm of the root with hair follicles with scissors and put them into a 1.5 mL centrifuge tube; S2. Digestion of hair follicles: Add 380 μL of tissue extract and 20 μL of proteinase K to the above centrifuge tube, mix thoroughly and centrifuge to fully immerse the hair follicles, and digest in a 56°C water bath for 1-2 hours. Flick the sample with your fingers to accelerate the digestion process. S3, DNA extraction: first, add 400 μL of Tris-saturated phenol to the centrifuge tube obtained in S2, mix by inversion for 3 min, and centrifuge at 12000 g for 5 min at 4°C; then take the supernatant and put it into another new 1.5 mL centrifuge tube, add 400 μL of a mixture of Tris-saturated phenol: chloroform: isoamyl alcohol = 25:24:1, mix by inversion for 3 min, and centrifuge at 12000 g for 5 min at 4°C; then take the supernatant and put it into another new 1.5 mL centrifuge tube; S4, DNA precipitation: add 360 μL of isopropanol and 40 μL of 10 M ammonium acetate to the centrifuge tube obtained in S3, mix by inversion for 3 min, centrifuge at 12000 g for 5 min at 4°C to precipitate the DNA to the bottom of the centrifuge tube, and discard the supernatant; then add 2 times the volume of the supernatant to the centrifuge tube with -20°C pre-cooled ice ethanol, invert the centrifuge tube for 2-3 min, centrifuge at 12000 g / min for 4 min, and discard the supernatant; S5. Washing: Rinse the DNA precipitate in the centrifuge tube obtained in S4 twice with 70% ethanol, centrifuge at 12000g for 4 minutes each time, use a pipette to absorb the residual ethanol in the centrifuge tube, open the centrifuge tube and place it under a fume hood for 10 minutes until the white precipitate in the tube turns into transparent particles, add an appropriate amount of TE buffer to dissolve the DNA, mix it by blowing with a pipette tip, and then store it in a -20℃ refrigerator for long-term storage.

3. The method for efficiently extracting pig hair DNA according to claim 2, characterized in that: The tissue extract is prepared by mixing 50 mmol / L Tris-CL at pH 8.0, 100 mmol / L EDTA at pH 8.0, 100 mmol / L NaCL, and 1% SDS.

4. The method for efficiently extracting pig hair DNA according to claim 1, characterized in that: The pig hair comes from the back of a pig, is collected along the direction of the hair, retains the hair with hair follicles, and is dried and stored in a sealed bag at room temperature.

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