KR20010016520A - Development of PCR-SSCP method for Identification of Hanwoo Meat - Google Patents

Abstract

PURPOSE: Provided is a new technique of polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) to identify accurately Korean beef cattle in a short time by removing processes which requires expensive restriction enzymes. CONSTITUTION: Polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) method comprises the following steps of: i) heating at 95 deg.C for 5 minutes MC1R genes multiplied by MC1R gene specific primer 1(forward primer: 5' - CAAGAACCGCAACCTGCACT-3' and reverse primer: 5'-CAAGAACCGCAACCTGCACT-3' and primer 2(forward primer:5'-CAAGAACCGCAACCTGCACT-3' and reverse primer:5'-CCAACGAGGCAGCAGGAT-3'; ii) separating an SSCP band through 12-15 % of unaltered polyacrylamide gel; and iii) dyeing the band with silver.

Description

Development of Polymerase Chain Reaction-Single-stranded Polymorphism Gene for Identification of Hanwoo Beef {Development of PCR-SSCP method for Identification of Hanwoo Meat}

In order to enhance the international competitiveness of Korean cattle industry and induce the production of high-quality Korean cattle beef in preparation for the opening of livestock imports, differentiation strategies from imported beef or domestic dairy cattle are emerging as a very important concern. However, in the domestic beef distribution process, inexpensive foreign imported beef and cow meat are sold as high-quality beef cattle, leading to enormous economic loss and damage to consumers and livestock farmers as well as serious social problems. It is pointed out. According to the Ministry of Agriculture and Forestry's national audit data, consumers reported damages of about KRW 49.8 billion from sales of cows and beef cattle from 1998 to this year.

In particular, when live cattle imported into Korea were brought into Korea and slaughtered after six months of breeding in 2001, the domestic beef cattle were allowed to be distributed as domestic beef. Insight Research recently surveyed 100 Korean meat farmers in Seoul to confirm the image and competitiveness of Korean beef. The amount of Korean beef sold in Korean beef was 36.2% of the total. Scams were bought with 47% of consumers per 10 people, and nearly half of the consumers were fooled into buying Korean beef. It is causing the consequences. If the irregularity of distribution of beef packs is widespread, it is difficult for the Korean beef differentiation and origin labeling policies to be properly settled, and the weakening of the competitiveness of the Korean beef industry and beef distribution industry by weakening consumer distrust and spread of negative perceptions on Hanwoo meat. It is the cause. Therefore, to prevent the sale of Hanwoo beef bluffs, it will be necessary for the government to continuously monitor and supervise distribution, but when scientific and objective Hanwoo beef discrimination and diagnostic technology are supported, consumers' confidence in Hanwoo beef will be restored and confidence will be resolved. And quality differentiation with cow's meat may be correctly established. In addition, the westernization of dietary patterns has led to changes in the dietary structure, leading to increased livestock consumption and reduced grain consumption. In addition, domestic meat consumption per capita was 29.3 kg in 1997, 34.7 kg in 2001, and 2004. It is expected to increase to 37.3kg. However, the current self-sufficiency of pure Hanwoo beef is less than 40%, and more than 60% of domestically distributed beef is being served by cow and imported beef. Therefore, the development of the technology to distinguish Korean beef from Korean beef and domestic cows by identifying the unique genetic characteristics of Hanwoo as a native domestic animal at the DNA level is essential to the international competitiveness of domestic beef and to block the distribution of beef meat. This is a task that must be addressed to protect the farmers and the beef market.

Seeking, the outward characteristic of a cow, is the only object trait that can identify a breed to date and is one of the representative qualitative traits that can be used as a means of identifying individuals and breeds. However, even cattle breeds distinguished by their phenotypic phenotype are virtually impossible to identify if they are slaughtered and converted to beef.

Recently, due to the remarkable development of molecular genetics and genetic engineering technology, several researchers have reported the possibility of breeding according to species-specific DNA sequence differences at the DNA level, as well as breeding in the same species. In Korea as well, the development of technology specific to Korean cattle breeds for identifying Korean cattle, which is Korean's unique genetic gene, has been of interest and interest to many researchers. In particular, RAPD (random amplified polymorphic DNA) technique has been mainly used for the detection of Hanwoo specific DNA markers for the determination of Korean beef cattle, but the RAPD technique is highly sensitive to PCR amplification conditions and results. It has been pointed out that there are many problems in practical use because of low reproducibility and low accuracy of judgment. On the other hand, a technique for clearly distinguishing Hanwoo beef from Holstein cow meat and Angus imported meat has been developed by analyzing PCR-RFLP (restriction fragment length polymorphism) of the melanocortin 1 receptor (MC1R) gene involved in bovine color development. Et al., 2000; Kim et al., 2000).

This PCR-RFLP technique is evaluated as a reliable discrimination technique compared to the previously reported PCR-RAPD method. However, in order to detect the RFLP genotype, there must be a nucleotide sequence that can be recognized by a specific restriction enzyme. The analysis process is rather complicated and requires a long analysis time. In particular, when the Korean beef meat discrimination technology is industrially applied to carry out a large amount of samples in earnest and the related technologies are widely expanded and distributed, first of all, the simplicity of the discrimination technology and the promptness and accuracy of the determination result are required.

Therefore, PCR-A is a new method for discriminating Korean beef, with the aim of developing easier, faster, more accurate, more economical and more efficient Korean beef, while overcoming the problems and limitations of existing Korean beef. Single strand conformation polymorphisms (SSCP) have been developed.

II. Materials and methods

1. Materials

For the test materials used in this study, meat samples were collected from 100 Korean cattle and 50 dairy cattle at the slaughterhouse. In addition, ten cattle of Angus, Hereford, and Charolais three varieties were randomly selected as the imported beef bred at Daegwallyeong Branch of Livestock Technology Research Institute.

2. Experimental method

1) Isolation and Purification of Genomic DNA

Separation and purification of genomic DNA from blood (3 ml) and muscle tissue (10 g) was performed by some modifications of Miller et al. (1989), and the isolated DNA was purified by TE buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA). Dissolved. The DNA concentration of each sample was measured by UV adsorption at 260 nm with a spectrophotometor.

2) MC1R primer synthesis and PCR amplification

The MC1R gene is a primer for amplifying the E-locus 138bp fragment located at 318 to 455 of the bovine receptor BDF3 registered in GenBank (S71017). The forward primer is 5'-CAAGAACCGCAACCTGCACT-3 'and the reverse primer is 5 A primer for amplifying 390bp of '-GCCTGGGTGGC CAGGACA-3' and 318th to 707th sequencing region, the base primer of 5'-CAAGAACCGCAACCTGCACT-3 'and reverse primer of 5'-CCAACGAGGCACAGCAGGAT-3' are respectively designed. PCR amplification was performed using the GeneAmp PCR System 9600 (Perkin-Elmer Cetus, USA) under the following conditions. In other words, the reaction solution was adjusted to 10 μl of PCR by adding 20-40ng of template DNA, 0.5μM of primer, 200μM of dNTP, 10 × PCR buffer, and 1 unit of Taq DNA polymerase to 0.5ml tube. The PCR cycle was pre-heated for 5 minutes at 94 ° C, followed by 30 cycles of 1 minute at 94 ° C, 1 minute at 66 ° C, and 1 minute at 72 ° C. .

3) SSCP marker analysis

After amplification using two specific primer pairs containing the mutation region of the MC1R gene, 5 to 6-fold formamide dye (98% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05) was added to 3 μl of PCR amplification products. % xylen cyanol) was added. Each denatured solution was denatured at 95 ° C for 5 minutes, immediately stored in ice for 5 minutes to prevent reannealing, followed by electrophoresis using 12-15% unmodified polyacrylamide gel, and detection of SSCP DNA bands by silver staining. It was.

III. Results and Discussion

MC1R is a hormone receptor that stimulates melanin proliferation and synthesis in mammals and has been found to play an important role in groping expression. As a method for detecting mutations in the MC1R gene, PCR-RFLP spectroscopy is used in several animal species. There have been studies of color shift (Robbins et al., 1993; Klungland et al., 1995; Vage et al., 1997, 1999; Kijas et al., 1998; Kim et al., 2000; Lee et al., 2000; Chung et al., 2000). In this study, PCR-SSCP analysis was used to detect mutations in the MC1R gene faster and more accurately than the conventional PCR-RFLP method. The structural differences in electrophoresis between varieties were compared and analyzed. For SSCP analysis of the MC1R gene, two primers with 318-455 and 318-707 base sequences of the BDF3 cDNA region were synthesized and amplified, respectively, and then denatured with 138bp and 390bp amplified products, followed by non-denatured polyacrylamide gel. SSCP patterns of each variety are shown in Figures 1 and 2. That is, as shown in Figure 1, when 138 bp amplified product was electrophoresed, single base substitution (T → C) and deletion (G) were determined in the codons determining amino acids 99 and 104 according to the base substitution of the point mutation. ), Four genotypes of E ^D / E ^D , E ^D / e, E ⁺ / e and e / e were detected. In addition, the SSCP genotype of the 390bp amplification product including the 319-707 nucleotide sequence was compared to compare the SSCP separation ability according to the molecular weight of the PCR amplification product, as shown in <Figure 2>. Although there are single base substitution and deletion sites in amino acid region 99 and 104, only two haplotypes were detected due to the limitation of separation ability. These results can be interpreted as a phenomenon in which the amplification of the PCR amplification product is less than 200bp with a detection accuracy of 70-95%, but the separation ability is reduced to 50% or less when it is about 400bp or more (Michaud et al., 1992).

M Hanwoo Holstein Angus Hereford Charolais M

━━━━━━━ ━━━━━━━ ━━━━━━━━━━ ━━━━━ ━━━━━

^{1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30}

<Figure 1> Electrophoresis of the PCR-SSCP genotypes of the MC1R gene in Korean beef, Holstein dairy and imported beef varieties (Angus, Hairford and Charlore). In electrophoresis, 1, 2, 3, 4, 6 and 22 to 30 lanes are e / e genotypes, 5 lanes are E ⁺ / e genotypes, and 7, 9, 10, 12 and 13 to 21 lanes are E ^D / E ^D Genotypes and 8 and 11 lanes are E ^D / E ⁺ genotypes. M: Standard size marker.

Hanwoo Holstein Angus Hereford Charolais

━━━━━━━━━━ ━━━━━━━ ━━━━━━━ ━━━━━━ ━━━━━━━

〈Figure-2〉 Korean Beef, Holstein Beef and Imported Beef Breeds (Angus, Hairford, and Charore)

Electrophoresis image comparing PCR-SSCP markers of MC1R gene for.

As described above, PCR-SSCP genotypes of the MC1R gene detected using two PCR amplification products, 138bp and 390bp, were compared between Hanwoo, dairy and imported beef breeds, as shown in <Figure 1 and 2>. Variety-specific SSCP genotypes were identified, showing a clear difference between Hanwoo, Holstein and Angus species according to the mobility of single-strand substitution and deletion. In other words, in the result of detecting the SSCP genotype using a PCR amplification product of 138bp (Fig. 1), only two types of E ⁺ / e and e / e were detected in Korean cattle but E ^D / E ^D and E for Holstein cows. ^D / e and Angus species detected only the E ^D / E ^D genotype, which showed a clear difference in the appearance of SSCP genotype between varieties. In addition, the SSCP genotype was analyzed using a PCR amplification product of 390bp (Figure 2). As a result of relatively high molecular weight, it was difficult to determine detailed and accurate genotypes, but two bands of Hanwoo and one of Holstein cow and Angus species Only bands were detected, respectively, and a clear difference in SSCP band appearance was recognized. These results suggest that the 390bp amplification product can be a very practical method for the easy and rapid determination of Hanwoo beef by simply using DNA band patterns expressed on electrophoresis gels regardless of the detailed genotypes of multiple beef samples. do.

Therefore, the SSCP genotype of the groping-specific MC1R gene developed in this study can be used as a very useful DNA marker for distinguishing domestic and imported cows from Angus imported meat. Could be. The difference in the detection ability according to the mobility of SSCP band between Hanwoo, Holstein, and Angus species determines the substitution of single base (CTG (Leu) → CCT (Pro)) and amino acid 103 at the 99 base sequence of MC1R gene. In the codon, GGT (Gly) → GTG (Val) results in the deletion of a second G monobase, which is interpreted as being able to distinguish between different breeds of Hanwoo and Holstein and Angus (Klungland et al., 1995; Chung et al., 2000). .

As a result of analyzing the frequency of SSCP genotypes of MC1R loci in three varieties of Korean cattle, cows and imported beef, Hanwoo had two genotypes, E ⁺ / e and e / e. Among them, the e / e type showed a very high appearance rate of 89%, whereas the E ⁺ / e type had a low appearance rate of 11%. However, Holstein species having a look at the black spots do not emerge at all beef E ^D / E ^D and E ^D / e E ^D / E ^D-type two types genotype of a 96% and 4%, respectively, a very high prevalence In addition, single genus, Angus, was detected in E ^D / E ^D genotype not present in Korean cattle. As such, there is a significant difference in the frequency of MC1R genotypes among cattle breeds, and this result is interpreted as a genetic difference according to the groping phenotypic variation between breeds and also reflects the characteristics of the breed.

<Table 1> Appearance Frequency of Varieties of MC1R Genotypes Detected by PCR-SSCP Method

kind Number of samples MC1R genotype E ^D / E ^D E ^D / E ⁺ E ^D / e E ⁺ / E ⁺ E ⁺ / e e / e Hanwoo 100 11 (0.11) 89 (0.89) Holstein 50 47 (0.96) 3 (0.04) Angus 10 10 (1.00) Hereford 10 10 (1.00) Charolais 10 10 (1.00)

In the past, studies on the clear association between MC1R genes and various groping phenotypes in cattle breeds have not been carried out, but PCR-RFLP analysis using restriction enzymes that recognize specific sequencing patterns can be used. Three alleles, E ^D , E ⁺ and e, have been classified and six genotypes dominated by them have been reported (Klungland et al., 1995; Klungland and Vage, 1999). In addition, Joerg et al. (1996) analyzed the nucleotide sequence of the MSHR gene and found that Holstein species with red color can be distinguished from black color varieties due to deletion of specific base. In addition to bovine breeds, Kijas et al. (1998) reported that missense mutations were associated with chromosomal mutations by PCR-RFLP and SSCP methods to identify differences in base sequences of MC1R loci in pigs with different groping phenotypes. Marklund et al. (1996) also found that a single missense mutation was associated with a chestnut phenotype in the analysis of the nucleotide sequence of equine MSHR. Recently, Jeong et al. (2000), Kim et al. (2000) and Lee et al. (2000) used the MC1R gene by PCR-RFLP method using different specific primers and restriction enzymes to distinguish Hanwoo cattle from Holstein cows and Angus imported meat. And based on the frequency of occurrence of specific genotypes of certain breeds, Hanwoo has been able to reliably distinguish Hangus from Angus. Currently, there are many varieties of cattle bred all over the world and it may be theoretically impossible to distinguish all cattle breeds, but among the domestically grown Holstein cows and imported varieties, Angus species are black spots and blackish brownish brown cattle. Because of the radical difference from the Korean beef distribution process, the MC1R genotype is completely different from Korean beef because of the Holstein breed cows, which are the mainstream of the domestic beef market in the domestic beef distribution process, and the Angus variety, which occupy a high proportion of imported meat. Even when converted to beef form after slaughter, Korean beef can be clearly distinguished from dairy beef and Angus imported meat, so it can be used for quality certification and detection of illegal meat.

In particular, the PCR-SSCP genotype is well known as a highly sensitive DNA polymorphism detection method that can easily and quickly detect mutations that cannot be identified by the RFLP method (Blanche et al. 1997; Hennessy et al., 1998). ). Therefore, the PCR-SSCP marker of the MC1R gene developed in this study was compared to the conventional PCR-RFLP method using two types of restriction enzymes. As it has the advantage of directly detecting the polymorphism of mutation-induced genes quickly and accurately and easily, it can be said to be a method of discriminating Hanwoo meat, which has fastness, accuracy, economy, convenience and practicality. Therefore, the PCR-SSCP genotype of the MC1R gene can be used as a very efficient, economical and useful DNA marker to distinguish Hanwoo beef from dairy cows and Angus imported meat.

1. Field of invention

Biotechnology and Genetic Engineering-Animal Genetic Engineering

The technology developed by the team belongs to `` Development of PCR-SSCP Gene Identification Method for Korean Beef Meat, '' which is a field of genetic engineering. As a genetic identification method for the identification of varieties of Korean beef, imported beef and cow meat using DNA markers, the previously reported technology is RAPD and SCAR analysis technology. Recently, a new discrimination technology using PCR-RFLP marker has been reported. .

2. Prior art in the field

① Method of discriminating Korean beef meat using RAPD and SCAR marker

The PCR-RAPD (Random Amplified Polymorphic DNA) technique uses a random oligonucleotide primer (random primer) with a short nucleotide sequence (10 bp) to randomly amplify the DNA sequence region in the genome to detect a specific DNA band and breed it. It is a technique that attempts to distinguish the breed of cattle by using it as a specific RAPD marker. However, RAPD technology is not only very sensitive to PCR amplification but also very low in reproducibility and accuracy (Yu and Pauls, 1992; Ellsworth et al., 1993; Macpherson et al., 1993; Meunier and Grimont et al., 1993). Since there are many problems to use, it is difficult to guarantee the reliability of the determination result, so it is difficult to transfer to industrial practical technology.

Using RAPD technique, cloning the band showing the difference between Hanwoo and other varieties, analyzing the nucleotide sequence, and making a new primer to stabilize and amplify the gene, to obtain more desirable reproducibility of the technique. SCAR (sequence characterized amplified regions) technique is basically the same as PCR-RAPD technique, but after sequencing the nucleotide sequence for the species-specific band, the base is synthesized by adding 10bp of base from the end to the 10bp RAPD marker. As a method of detecting primers as SCAR primers, it can be said that the technique of reproducibility of RAPD is partially improved. However, this method also uses the PCR-RAPD technology, which has not been fully studied and verified about the SCAR marker for discriminating Korean beef.

② Method of discriminating Korean beef meat using PCR-RFLP marker

The recently developed PCR-RFLP marker using the PCR-RFLP marker is a method for discriminating Hanwoo beef, using the breed-specific genotype by RFLP analysis of MC1R gene involved in bovine color development. As a distinguishing technique, it is a method of discriminating Hanwoo beef with high accuracy and reproducibility and high reliability compared to the method using RAPD and SCAR marker. However, in order to detect the PCR-RFLP genotype, there must be a nucleotide sequence that can be recognized by a specific restriction enzyme. Therefore, there is a limitation that only a limited restriction enzyme recognition site can be detected. As a result, it is pointed out that the analysis process is rather complicated and takes a long time. Therefore, for the practical use and generalization of the Korean beef meat discrimination technology, it is necessary to develop a simple, fast and accurate method of discriminating technology, low cost of analysis, and economical Korean beef meat discrimination technology.

As described above, the problem of discrimination between Korean beef cattle, cow cattle, and imported cattle through RAPD marker has been performed by many researchers (Lee Chang-soo et al., 1994; Cho, Byeong-uk and Han, Jae-yong, 1994; , 1995; Jung Ryong Rong et al., 1995). However, RAPD technology is not only very sensitive to PCR amplification but also very low in reproducibility and accuracy (Yu and Pauls, 1992; Ellsworth et al., 1993; Macpherson et al., 1993; Meunier and Grimont et al., 1993). Because it contains many problems to use it, it is difficult to guarantee the reliability of the decision result and it is difficult to transfer to industrial practical technology.

In particular, due to the characteristics of the technology of RAPD technology, it is very difficult to obtain the same test result by reacting sensitively by many experiment factors such as experimenter, sample DNA, PCR reaction solution composition, PCR amplification condition (reaction temperature and number of cycles). It is lacking. In addition, in order to improve the very low reproducibility of RAPD technology, SCAR (sequence characterized amplified regions) markers using about 20 bp primer longer than 10 bp RAPD primer have been reported in pieces, but this method also uses PCR-RAPD technology. As of yet, sufficient research and verification of the SCAR marker for the determination of Korean beef has not been carried out, which has not been put into practical use.

On the other hand, as a recently developed gene identification technique, Hanwoo meat discrimination method using PCR-RFLP marker of MC1R gene related to cow groping clearly distinguishes Hanwoo, dairy cow and Angus species using allele-specific primer and two kinds of restriction enzymes. Therefore, it is evaluated as the test method with the highest accuracy and reliability of discrimination results among the Korean beef meat discrimination methods reported so far. However, only limited restriction enzyme recognition sites can be detected and restriction enzyme processing is required, which requires expensive restriction enzymes, complicated analysis process, and long analysis time. Therefore, the technical task of this research and development is more efficient than the previously reported methods, and can guarantee the speed and accuracy of analysis, low analysis cost and economics, and technical reproducibility, as well as technical originality, novelty and advancement. The purpose of this study is to develop a Korean beef cattle discrimination technology using the PCR-SSCP technique, which has excellent characteristics and advantages, and to industrially use them to establish a new technology for discriminating Korean beef cattle.

The advantages and excellence of this PCR-SSCP technology are compared with the existing methods and presented in <Table-2>.

〈Table-2〉 Comparison of Korean Beef Meat Identification Technique

Plate tassel PCR-RAPD PCR-RFLP PCR-SSCP Release method PCR amplification: 3-4 hours / 45 cycles Electrophoresis: 3-4 hours / 50 Volts Total analysis time: 6-8 hours PCR amplification: 2-3 hours / 30 cycles restriction enzyme treatment: 2-3 hours Electrophoresis: 1.5-2 hours / 50 Volts Total analysis time: 6-8 hours PCR amplification: 2-3 hours / 30 cycles Electrophoresis: 1.5-2 hours / 300 Volts Silver staining: 1 hour Total analysis time: 4.5-5 hours Economics medium Ha Prize accuracy Ha Prize Prize Reproducibility Ha Prize Prize responsibility Ha Prize Prize Promptness medium Ha Prize Simplicity Prize medium Prize

Conventional Korean cattle breed discrimination technology using RAPD marker or SCAR marker has high sensitivity to PCR amplification conditions and low reliability of analysis results and reliability. I can't. In addition, the PCR-RFLP marker has a higher accuracy and reproducibility than the RAPD marker, but it has a disadvantage in that the analysis process is inconvenient and takes a long time due to the restriction enzyme treatment process. However, the new Hanwoo meat discrimination technology developed by the researchers is an advanced Hanwoo gene identification technology using Melanocortin 1 receptor (MC1R) gene, which is involved in the development of cattle. After amplification, add 5-6 fold formamide dye (98% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylen cyanol) to 3µl of PCR amplification products (138bp or 390bp), and centrifuge at 95 ℃. After denaturation for 5 minutes, immediately stored in ice for 5 minutes to prevent reannealing, then 10-15% non-denaturing polyacrylamide gel (7.5 ml of 40% Acryiamide solution, 5 ml of 10X TBE buffer, 170 µl of 10% Ammonium persulfate, 70 µl) After electrophoresis using TEMED), DNA band was expressed by silver staining method, and distinction between Hanwoo beef, domestic cow meat and Angus species imported meat was made by using varieties specific SSCP genotype as DNA marker. The.

Details on the principle of operation, composition, and analysis method of the Hanwoo beef gene identification technique are as follows.

〈# Figure-3. Diagram of Korean Beef Meat Discrimination Technology Using PCR-SSCP Technique

Beef meat

↓

Genomic DNA Isolation

↓

Specific primer synthesis and PCR amplification

↓

SSCP marker analysis

↓

Differentiation of Korean Beef (Different from Holstein and Beef)

end. method of inspection

1) Material Preparation: Raw meat or blood

2) Reagent-DNA Extraction Solution

-MC1R gene specific primer:

Primer 1 (138bp amplification) forward primer: 5'-CAAGAACCGCAACCTGCACT-3 '

reverse primer: 5'-GCCTGGGTGGCCAGGACA-3 '

Primer 2 (for 390bp amplification) forward primer: 5'-CAAGAACCGCAACCTGCACT-3 '

reverse primer: 5'-CCAACGAGGCACAGCAGGAT-3 '

Taq DNA polymerase

10X PCR buffer, 500mM KCl, 100mM Tris-HCl (pH 9.0) 15mM MgCl2

dNTP: 25mM dATP, dCTP, dGTP, dTTP mixture

Instrument-Spectrophotometer, Thermal cycler (DNA amplifier)

2) method

① DNA Extraction: Extraction and purification of genomic DNA from 5g of raw meat samples were extracted by the general separation and purification method. The collected DNA was dissolved in TE buffer, and the extracted DNA concentration was measured by UV absorption at 260nm with spectrophotometor. do.

② DNA amplification: The extracted DNA (50-100ng) is mixed in a 20ul tube containing the following reagent (DNA amplification reaction solution) and perform DNA amplification under the following PCR reaction conditions.

* DNA amplification reaction solution 10X PCR buffer 10X dNTP Primer (50ng / μl) Taq DNA polymerase dH2O 5 μl 5 μl 1 μl 1 μl 8 μl * PCR reaction conditions Predenaturing Denaturing Annealing Extension Final-extension 94 ° C / 5 min 94 ° C / 1 min 66 ° C / 1 min 72 ° C / 1 min 72 ° C / 5 min Total 20 μl 30Cycles

③ SSCP analysis: 5 ml of formamide dye (98% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylen cyanol) was added to 3 μl of PCR amplification product. After 5 minutes on ice, electrophoresis was performed using 12-15% unmodified polyacrylamide gel, and then stained with silver.

④ DNA detection: After electrophoresis on 12-15% unmodified polyacrylamide gel, silver staining is used to detect SSCP band.

1) Hanwoo meat discrimination technology using the PCR-SSCP analysis method of the groping-related MC1R gene developed by the researchers is more stable and accurate than the conventional RAPD or SCAR marker technology. Hanwoo meat gene identification technology combines rapid, accurate, economical, convenience and practicality with the advantage of easily and quickly and accurately detecting the polymorphism of a gene caused by mutation without using enzyme.

2) This PCR-SSCP technology is a new method for discriminating Korean beef, which can be used more efficiently and industrially. It is possible to quickly and easily distinguish between Korean beef, domestic cows, and Angus. It can be used as inspection technology for crackdown, and at the same time, it can contribute to income increase and consumer protection of Korean beef cattle farmers by inducing quality differentiation such as quality certification of beef raw meat products.

3) This gene identification technology provides objective and scientific indicators for Korean beef, which can fundamentally solve the problems of illegal illegal distribution system such as Hanwoo beef thickened phenomenon, thereby creating domestic demand and quality competitiveness of Korean beef through high quality meat production. It can strengthen the international competitiveness of domestic Korean beef industry.

4) The science of raw meat that can add value and differentiate from cheap beef and imported beef by maximizing the policy aspect of producing and supplying high quality animal protein in Korea for stable national health and the consumption characteristics of Korean beef. The objective and objective breeding discrimination technique will contribute to relieving the distrust of consumers and securing the reliability of Korean beef.

Claims (1)

MC1R gene specific primer 1 (forward primer: 5'-CAAGAACCGCAACCTGCACT-3 'and reverse primer: 5'-GCCTGGGTGGCCAGGACA-3') and primer 2 (forward primer: 5'-CAAGAACCGCAACCTGCACT-3 'and reverse primer: 5'- PCR comprising the process of heat-modifying the MC1R gene product amplified by CCAACGAGGCACAGCAGGAT-3 '), separating SSCP bands using a 12-15% unmodified polyacrylamide gel, and then staining with silver. -Method of Korean Beef Meat by SSCP Gene Analysis