KR20030064002A - Amplification of DNA Fragment Using Swine Single Hair Follicles for Cloning or Genotyping of the DNA Fragment - Google Patents

Abstract

PURPOSE: A method for amplification of a DNA fragment using swine single hair follicies for cloning or genotyping of the DNA fragment is provided, thereby easily and accurately amplifying the DNA fragment of swine without conferring stresses to pigs. CONSTITUTION: A method for amplification of a DNA fragment using swine single hair follicies for cloning or genotyping of the DNA fragment comprises the steps of: first amplifying the DNA fragment extracted from the swine single hair follicies by primary PCR; and second amplifying the first amplified DNA fragment by nested PCR using primers used in the primary PCR, primers having lower concentration than that of dNTPs and dNTPs, wherein booster PCR may be optionally carried out after the second amplification process; the primers used in the primary PCR and booster PCR are P1 and P2; the primers used in the nested PCR are P3 and P4; the concentration of primers in the primary PCR is 0.003 to 0.3 pmole; the concentration of dNTPs in the primary PCR is 0.002 to 0.2 mM; the concentration of primers in the booster PCR is 0.03 to 3 pmole; the concentration of dNTPs in the booster PCR is 0.02 to 2 mM; the concentration of primers in the nested PCR is 0.3 to 30 pmole; and the concentration of dNTPs in the nested PCR is 0.2 to 20 mM.

Description

Amplification of DNA Fragment Using Swine Single Hair Follicles for Cloning or Genotyping of the DNA Fragment}

According to the present invention, a genetic DNA detection is performed after cloning specific DNA fragments of pigs using a pig hair or treating the identified DNA fragments with a restriction enzyme. A method for amplifying a specific DNA fragment in a sufficient amount that can be determined from.

Specifically, the present invention uses a genome extracted from the hair follicle of a very small amount of genome (genome) to identify a specific DNA fragment of the pig or a DNA through a polymerase chain reaction (PCR) polymerase chain reaction (PCR) The present invention relates to a method of amplifying a specific DNA fragment in a sufficient amount to detect a genetic variation of the DNA fragment in a gel by treating the fragment with a restriction enzyme and performing electrophoresis.

Polymerase chain reaction is a technique for amplifying DNA fragments at specific sites in a genome using polymerases, primers, dNTPs, and templates. However, when amplifying a specific DNA fragment through a polymerase chain reaction from a genome extracted from a sample having a very low content of the genome, in particular, a partially degraded genome, the specific DNA fragment of interest is not amplified or amplified. DNA mutations are amplified and genetic variation testing using them results in inaccurate test results. Even when a specific DNA fragment of interest is amplified, the amount of DNA fragments amplified is extremely small, so that the genetic Mutation testing becomes impossible (Lee et al., Journal of Forensic Science, 1991, Volume 36 Lung 320-330, Han et al., Nucleic Acids Research, 1992, Volume 20 Lung 6419-6420).

In particular, when the desired DNA fragment is present as a single DNA fragment in the genome, the rate at which artifacts that are not amplified or nonspecifically a large number of nonspecific DNA fragments are amplified compared to repeatable DNA fragments. And the probability is higher that genetic testing is impossible.

Genetic traits of pigs are very important economic traits for breeding and breeding pigs, and the testing of these traits is very important both in academic and economic terms.

The lyanodyne receptor coding gene is the most representative gene for genetic testing in pigs, and it is a very important economic trait in pigs as a unique sequence.A mutant recessive gene exists in homozygous (n / n) form. In this case, the pig is very sensitive to stress and dies under severe stress, and the meat is very bad during slaughter, so that the flesh is pale, soft and produces juicy PSE pork. This will cause enormous damage to pig farmers. Therefore, the whole world is devoted to eliminating the above-mentioned recessive gene mutant gene from pigs.

In general, genetic variation investigation through polymerase chain reaction in livestock uses genomes extracted from blood or ear tissue (Fujii et al., Science, 1991, Volume 253, pp. 448-451, Hughes et al., Journal of Animal Breeding and Genetics, 109 pages 109 pages 465-476, Voeli et al., Animal Genetics, pages 25 pages 1994-59 59-65, Nakajima et al., Journal of Animal Science, pages 74 pages 1996 2904-2906).

In the investigation of such genetic variation, a sample for extracting a dielectric is very important. That is, in the case of pigs, a large amount of intact intact genome can be separated and extracted from pig blood or ear tissue, which is generally used as a sample, but the collection of blood or ear tissue adds stress to the pig and causes the pig to die. The development of a way to solve these problems is very important because it causes a lot of inconvenience to the pig farmers, such as causing a large loss to the pig farming farms by temporarily or temporarily stopping the weighting effect. All.

Accordingly, an object of the present invention is to use a pig's hair root which can be collected without stressing the pig instead of the existing ear tissue or blood that stresses the pig and temporarily stops growth and weighting economically. It provides a method for identifying DNA fragments containing genes.

It is also an object of the present invention to provide a method for amplifying a genetic variation of a DNA fragment comprising a gene identified by this method in a sufficient amount to be examined in a gel after restriction enzyme treatment.

Figure 1 is an electrophoresis picture after the polymerase chain reaction and restriction enzyme treatment for the genome extracted from unfresh pig hair roots.

<Brief description of the main parts of the drawing>

1.molecular size marker,

2. normal genotype pigs (N / N),

3. heterozygous genotype pigs (N / n),

4. recessive genotype pig (n / n).

The present inventors have conducted a number of studies to achieve this purpose. As a result, a very small amount of the genome present in the sample is partially degraded, and as a result, a single genome is extracted from pig hair roots having a very small amount of available genome in the polymerase chain reaction. Efficiently amplify DNA fragments, or identify and identify the DNA fragments identified in the gel by UV irradiation after staining with ethidium bromide. The present invention has been completed by developing a method for amplifying DNA fragments in a sufficient amount.

As described above, the present invention is a method for identifying a specific DNA fragment of a pig using a pig hair or treating the identified DNA fragment with a restriction enzyme and then amplifying the specific DNA fragment in an amount sufficient to determine the genetic variation detection in the gel. It is about.

Specifically, the present invention uses a genome extracted from the hair follicle of a very small amount of genome (genome) to identify a specific DNA fragment of the pig or a DNA through a polymerase chain reaction (PCR) polymerase chain reaction (PCR) The present invention relates to a method of amplifying a specific DNA fragment in a sufficient amount to detect a genetic variation of the DNA fragment in a gel by treating the fragment with a restriction enzyme and performing electrophoresis.

In particular, the pig hair roots delivered by mail have been reported to have a much smaller genome compared to fresh hair roots. That is, since a large amount of the genome existing in the old hair root is already decomposed and cannot be used for the polymerase chain reaction, the genome extracted from the old hair root has a relatively small amount of the available genome compared to the fresh hair root. DNA fragmentation amplification is difficult. However, the use of these old hair roots eliminates the need for the inspector to visit the pig farm in person, and also allows for testing of samples delivered by mail to test for specific DNA fragments or genetic variation of identified DNA fragments in a complete laboratory. It's a very useful way.

In the present invention, the gene encoding the lyanodyne receptor, which is a representative gene for genetic testing of pigs, as the DNA fragment to be tested in the identification of specific DNA fragments using the porcine hair follicle and the development of a genetic variation test method of the identified DNA fragments ( swine ryanodine receptor gene) was selected.

The method according to the present invention uses a genome extracted from pig hair roots and performs a two or three step polymerase chain reaction. First, the primary primary PCR is performed within the primer concentration of 0.003 pmole to 0.3 pmole and the dNTPs concentration of 0.002 mM to 0.2 mM, and in the secondary PCR, a booster PCR using the same primer as the primer used for the primary PCR (booster PCR), wherein the concentration of the primer and dNTPs is 10 times the concentration used for the first PCR, that is, PCR is performed within the range of primer concentrations of 0.03 pmole to 3 pmole and dNTPs concentration of 0.02 mM to 2 mM. do. Tertiary PCR is a nested PCR using a primer having a base sequence different from the primary or secondary PCR, wherein the primer is 0.3 pmole to 100 pmole, dNTPs in the concentration range of 0.2 mM to 20 mM PCR is carried out.

These three rounds of PCR (primary PCR, booster PCR, nested PCR) and two rounds of PCR (primary PCR, nested PCR) are characterized by very low concentrations of primers and dNTPs in primary PCR (0.003). pmole-0.2 pmole) is very important.

In the PCR of the present invention, a primer of a gene encoding porcine lyanodyne receptor is used.

Specifically, a pair of primers (P1 and P2) having the following sequences are used for the primary PCR and the booster PCR.

P1: 5'-TCCAGTTTGCCACAGGTCCTACCA-3 ',

P2: 5'-ATTCACCGGAGTGGAGTCTCTGAG-3 '.

The P1 primer corresponds to the base sequence of 493 to 470th base from the mutant base (base # 433) of the lyanodyne receptor gene (5 'upstream), and the P2 primer is the mutant base of the lyanodyne receptor gene. (3 'downstream) corresponds to the base sequence from the 141st base to the 164th base.

Therefore, when the polymerase chain reaction is carried out using a primer having such a nucleotide sequence, it contains the 1843 base, which is the mutant base position causing the bite, and the length of the DNA fragment to be amplified is 659 bases.

Nested PCR uses a different pair of primers (P3, P4), and their nucleotide sequences are as follows.

P3: 5'-CATGTATGGACAACATCCACCT-3 ',

P4: 5'-CTGGTGACATAGTTGATGAGGT-3 '.

The P3 primer is the nucleotide sequence from base 416 to base 437 from the mutant base of the lyanodyne receptor gene (5 'upstream), and the P4 primer is the bottom (3' downstream) from the mutant base of the lyanodyne receptor gene. The base sequence is from the 64th base to the 85th base.

Therefore, when the polymerase chain reaction is carried out using a primer having such a nucleotide sequence, it contains the 1843 base, which is the mutant base position causing the bite, and the length of the DNA fragment to be amplified is 522 bases, which is P1. And a portion of DNA fragments amplified using P2 primers (Vogeli et al., 1994, Animal Genetics, Volume 25 Lung 59-65, Nakajima et al., 1996, Journal of Animal Science, Volume 74 Lung 2904-2906). .

As described above, the amplification products obtained by performing nested PCR are subjected to restriction enzyme treatment, followed by electrophoresis on an agarose gel to perform genetic mutation test, thereby amplifying the target gene fragments. And through this it is possible to determine the genotype of the test pig.

Using the amplification method of the present invention, amplification of the genome extracted from the hair roots of pigs after a period of time after extraction resulted in three types of genes encoding lyanodyne receptor, that is, one pair of normal lyanodyne receptor genes ( Swine with N / N), heterozygous genotype (N / n) pig with one normal lyanodyne receptor gene and one mutant recessive gene, and a recessive genotype (n) with both mutant recessive lyanodyne receptor genes / n) was found to exhibit the same amplification effect as when amplifying a genome extracted from fresh hair roots, blood and muscle. Therefore, according to the present invention, a specific gene is used by using a pig's hair root which can be collected without stressing the pig instead of the existing ear tissue or blood, which stresses the pig and temporarily stops growth and growth. It is possible to amplify a DNA fragment comprising a.

The method of amplifying certain DNA fragments according to the invention is particularly useful for amplifying DNA fragments in animals with hair on the skin, preferably in animals associated with animal husbandry.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

<Example 1>

Genome Extraction from Fresh Roots of Pigs and Fresh Roots Delivered by Mail

A 0.5 centimeter long hair was cut from the pig's hair root and washed once with distilled water. 0.5 centimeters of hair, including the hair root, were extracted with a dielectric extract buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 100 mM NaCl, 39 mM DTT, 200 ug / ml proteinase K, 2). % SDS) and then incubated for 12 hours at 55 ℃. After incubation, the same amount of phenol was added, followed by turbid turbidity, and centrifugation to recover the supernatant. The recovered supernatant was mixed with the same amount of chloroform, then turbidly washed and centrifuged to recover the supernatant.

2.5 times of ethyl alcohol, 1/10 times of 3.5M sodium acetate and pH 5.2 were added to the recovered supernatant and mixed. The mixture was allowed to stand at -70 ° C for 2 hours, and then centrifuged to recover the DNA pellet. The recovered precipitate was washed with 70% alcohol. After the precipitate was dissolved in distilled water, a dielectric was separated and purified using a commercially available silica bead product (GENECLEAN II KIT, BIO 101 Inc, CA, USA) and used for the polymerase reaction.

<Example 2>

1. Primary PCR

By the method of Example 1, a genome extracted from 2-5 unfresh hair roots stored at room temperature for 3 days after being delivered by mail from pig farms in Jeju, Chungcheong, and Gyeongsang-do areas was used as a template. The components are as follows: polymerase buffer (10 mM Tris-HCl, pH 9.0.50 mM KCl, 0.1% Triton X-100), 1.0 mM MgCl ₂ , 0.03 pmole P1 and P2 primers, 2.5 units, respectively. Taq enzyme, 0.02 mM dNTPs (dATP, dCTP, dGTP, dTTP).

PCR conditions for the polymerase chain reaction were repeated 5 minutes at 95 ° C for 1 minute (denaturation), at 94 ° C for 1 minute (denaturation), at 53 ° C for 30 seconds (annealing), and at 72 ° C for 1 minute (extension). It was.

2. Booster PCR

As a template, 5 ul of the primary PCR product was used. The same polymerase buffer as the primary PCR was mixed with 0.3 pmole of P1 and P2 primers, 0.2 mM dNTPs, 1.0 mM MgCl ₂ , and 2.5 units of polymerase, respectively. Then, PCR was performed under the same PCR conditions as the primary PCR.

3. Nested polymease chain reaction and DNA fragment identification

As a template, 5 ul of the booster PCR product was used, and the same polymerase buffer as the primary PCR was mixed with 3 pmole of P3 and P4 primers, 2 mM dNTPs, 1.0 mM MgCl ₂ , and 2.5 units of fryer. DNA fragments of the lyanodyne receptor gene were identified by carrying out the same PCR conditions as the head PCR.

4. Restriction Enzyme Treatment of Nested PCR Products

15 ul of the nested PCR product was treated with restriction enzyme CfoI at 37 ° C for 12 hours.

5. Electrophoresis

After restriction enzyme treatment, electrophoresis was performed on 1.5% agarose gel to examine genetic variation.

Figure 1 is an electrophoresis picture showing the results of the identification and genetic variation of the stress gene that causes the bite by the three times the polymerase chain reaction and restriction enzyme treatment after extracting the genome from the fresh pig hair root. In Figure 1, 1 is a DNA size marker (Molecular size marker), 2 is a normal genotype pig (N / N), 3 is a heterozygous genotype pig (N / n), 4 is a recessive gene type pig (n / n) The bands appearing characteristically.

As can be seen in Figure 1, a pig (N / N) having a normal pair of lyanodyne receptor genes is cleaved upon CfoI restriction enzymes to generate DNA fragments having 439 bases and DNA fragments having 83 bases. DNA fragments with 522 bases were not detected.

On the other hand, in the heterozygous genotype (N / n) having one normal gene and one mutant recessive gene, DNA fragments having 522 bases and DNA fragments having 439 bases recognized and cut by CfoI restriction enzyme and 83 It was detected that DNA fragments with bases were produced. In recessive genotype (n / n) pigs, only DNA fragments with 522 bases were detected.

The band of DNA fragments appearing on the gel in these experiments demonstrates that the desired DNA fragments have been identified, and the amount of specific DNA fragments identified is amplified so sufficiently that it is inconvenient to investigate genetic variation testing during restriction enzyme treatment. There was no sign.

<Example 3>

By the method of Example 1, using a genome extracted from fresh 2-5 hair roots, amplification by the same method as in Example 2 (primary PCR, booster PCR, nested PCR), and then the CfoI restriction of the same method As a result of enzymatic treatment and electrophoresis, the same result as in Example 2 using a dielectric extracted from unfresh hair roots was obtained.

<Example 4>

1. Primary PCR

By the method of Example 1, a genome extracted from 2-5 unfresh hair roots stored at room temperature for 3 days after being delivered by mail from pig farms in Jeju, Chungcheong, and Gyeongsang-do areas was used as a template. The components are as follows: polymerase buffer (10 mM Tris-HCl, pH 9.0.50 mM KCl, 0.1% Triton X-100), 1.0 mM MgCl ₂ , 0.03 pmole of P1 and P2 primer, 2.5 units, respectively. Taq enzyme, 0.02 mM dNTPs (dATP, dCTP, dGTP, dTTP).

PCR conditions, which are polymerase chain reactions, were denatured at 95 ° C for 5 minutes (denaturation) at 94 ° C for 1 minute (denaturation), at 53 ° C for 30 seconds (annealing) and at 72 ° C for 1 minute (extension) at 25 times. Repeated.

2. Nested PCR

Nested PCR was performed without booster PCR. In other words, 5 ul of the primary PCR product was used as a template, and the same polymerase buffer as the primary PCR was mixed with 3 pmole of P3 and P4 primers, 2 mM dNTPs, 1.0 mM MgCl ₂ , and 2.5 units of polymerase. Then, PCR was performed under the same PCR conditions as the primary PCR.

3. Restriction Enzyme Treatment and Electrophoresis

As a result of performing genetic tests on the nested PCR product using the same method as described in Example 2, the same result as in Example 2 was obtained.

Example 5

By the method of Example 1, using the genome extracted from the fresh 2-5 hair roots, the same method as in Example 4 was carried out to check whether the genetic variation was examined and the same results as in Example 4.

As can be seen from the above description, the amplification method of the present invention showed a perfect effect on the identification of specific DNA fragments using porcine hair roots or the genetic variation of the identified fragments. The method of the present invention using the pig hair root of the present invention, unlike the conventional DNA fragment identification or genetic variation test method using the blood and ear tissue samples of pigs, and without taking a hair root sample without stress from the pig The ability to amplify specific DNA fragments will greatly contribute to the identification of DNA fragments from the genome and testing for genetic variation.

Claims (10)

The genome extracted from porcine hair roots was amplified by primary PCR, and then again amplified by nested PCR using primers and dNTPs at concentrations lower than those of the primers and dNTPs used in the primary PCR. A method for amplifying a pig DNA fragment in an amount sufficient for identification or genetic variation testing of the pig DNA fragment. The method of claim 1, further comprising booster PCR followed by primary PCR. The method according to claim 1 or 2, wherein the primers used for the primary PCR and the booster PCR are primers P1 and P2 having the following sequences. P1: 5'-TCCAGTTTGCCACAGGTCCTACCA-3 ', P2: 5'-ATTCACCGGAGTGGAGTCTCTGAG-3 '. The method according to claim 1 or 2, wherein the primers used for the nested PCR are primers P3 and P4 having the following sequences. P3: 5'-CATGTATGGACAACATCCACCT-3 ', P4: 5'-CTGGTGACATAGTTGATGAGGT-3 '. The method of claim 1 or 2, wherein the concentration of the primer in the primary PCR is 0.003 pmole to 0.3 pmole. The method according to claim 1 or 2, wherein the concentration of dNTPs in the primary PCR is 0.002 mM to 0.2 mM. The method of claim 2, wherein the concentration of the primers in the booster PCR is 0.03 pmole to 3 pmole. The method of claim 2, wherein the concentration of dNTPs in the booster PCR is 0.02 mM to 2 mM. The method of claim 1 or 2, wherein the concentration of the primers in the nested PCR is 0.3 pmole to 30 pmole. The method of claim 1 or 2, wherein the concentration of dNTPs in the nested PCR is 0.2 mM to 20 mM.