KR100898721B1 - Method of identification between Hanwoo and foreign beef meats - Google Patents

Abstract

The method for discriminating Korean cattle and foreign introduced cattle according to the present invention is performed using Restriction Fragment Length Polymorphism (RFLP) of a sex-determining region Y (SRY) gene. The SL Wai gene is a gene of Bos taurus varieties, and includes a single nucleotide polymorphism (SNP) including a portion in which the 1707 th T base is mutated to the G base. Specifically, the method for discriminating the domestic and foreign introduced cows, the step of collecting DNA from the domestic and foreign introduced cows, the step of combining each of the DNA and the forward and reverse primers of the SAL Y gene, the binding product PCR amplification step, and the amplification product is cleaved with Mse I restriction enzyme and then analyzing the polymorphism comprises the step of classifying the genotype of the Korean cattle and foreign introduced cattle. According to the determination method, Bos taurus- derived From beef cattle and boss indices Foreign Brahmans can be accurately determined at the DNA level.

Description

Method of identification between Hanwoo and foreign beef meats}

1 is a bovine ( Bos taurus , AB039748) and foreign introductions ( Bos indicus , AY079145) is a sequence diagram showing the SRY gene sequence.

FIG. 2 is a conceptual diagram showing the size of specific nucleotide sequence recognition sites and DNA fragments of Mse I restriction enzyme.

3 is a bos taurus ( Bos The taurus) breeds of beef carcass and boss indicator (Bos indicus) amplifying the SRY gene of Brahman (Brahman) species varieties by PCR is a photograph showing a result of electrophoresis.

Figure 4 is a photograph showing the results of electrophoresis of the cleaved DNA fragment after RFLP treatment of Hanwoo and Brahman PCR amplification products.

The present invention relates to a method for discriminating Korean beef and foreign imported beef, and more specifically, Bos taurus ( Bos taurus) varieties of hanwooyuk and boss indicator carcass (Bos indicus) breed of Brahman (Brahman) species, and DNA displays factors can determine the hybridization varieties and Brahmin species (DNA marker) determination of hanwooyuk and foreign beef using method It is about.

In general, even cattle breeds that are distinguished by phenotypes such as groping, body shape, etc., which are external features, are almost impossible to identify after breeding beef. In the case of Korean beef, many cases of foreign imports of domestically imported beef and domestic cow meat are illegally distributed as Korean beef, which has been pointed out as a cause of damage to consumers and Korean cattle farmers as well as weakening the competitiveness of Korean beef industry. When cow beef is Hanwoo beef, 100% discriminating technology is secured and applied to the field. However, the technology that can be applied directly to the imported beef has not been developed yet.

An object of the present invention is to compensate for the shortcomings of the prior art as described above, Bos Taurus ( Bos taurus) varieties of hanwooyuk and boss indicator carcass (Bos indicus) breed of Brahman (Brahman) species and identify Brahmin hybrid DNA display factor (DNA marker) can be determined, and by using this effective determination of hanwooyuk and foreign introduced six To provide a method. In particular, the present invention focuses on the genetic fact that a lot of infusions of Brahmans , which are Bos indicus, in the case of non-introduced cows and mixed cows that are not purebred.

The method for discriminating Korean cattle and foreign introduced cattle according to an aspect of the present invention is performed using restriction fragment length polymorphism (RFLP) of a sex-determining region Y (SRY) gene. The Hanwoo is from Bos taurus and the foreign introduced cow is Brahman from Bos indicus.

The SL Wai gene is a gene of Bos taurus varieties, and includes a single nucleotide polymorphism (SNP) including a portion in which the 1707 th T base is mutated to the G base.

Specifically, the method for discriminating the domestic and foreign introduced cows, the step of collecting DNA from the domestic and foreign introduced cows, the step of combining each of the DNA and the forward and reverse primers of the SAL Y gene, the binding product PCR amplification step, and the amplification product is cleaved with Mse I restriction enzyme and then analyzing the polymorphism comprises the step of classifying the genotype of the Korean cattle and foreign introduced cattle.

The Hanwoo had two restriction enzyme fragment length polymorphisms and the foreign introduced cow had three restriction enzyme fragment length polymorphisms.

Hereinafter, the present invention will be described in detail.

Korean beef meat according to the present invention ( Bos taurus ) and exotic beef ( Bos indicus ) method includes restriction fragment length polymorphism (RFLP) of SRY SNP. The discrimination method is based on the bovine sequence registered in the GenBank using the RFLP, Hanwoo (AB039748) and Bos indicus ( Bos taurus ) varieties indicus ) , which analyzes structural differences in SRY gene sequences of Brahman, Brahman, and recognizes and cleaves specific sequences (T / TAA) with Mse I restriction enzymes after gene amplification (PCR) to analyze polymorphism of fragments. Steps.

Restriction enzyme fragment length polymorphism ( RFLP )

Restriction Fragment Length Polymorphism (RFLP) is a type of polymorphism found in most prokaryotic cells. It is a polymorphism of DNA that appears in varying lengths of fragments. This point mutation occurs in the nucleotide sequence recognized by the restriction enzyme, and the recognition site of the restriction enzyme is newly generated or the existing restriction enzyme recognition site is lost, thereby changing the DNA fragmentation pattern by the restriction enzyme, which is easily detected by electrophoresis. Can be. Since the polymorphism of DNA is different from person to person, RFLP has been applied to individual identification and forensic use as well as markers related to genetic diseases.

According to the discrimination method according to the present invention, it is possible to discriminate varieties in the same species by using the structural difference in the DNA sequence. That unique genetic resources of the country and the boss Taurus (Bos Taurus), one of the beef carcass Indy boss of the breed (Bos a method using a indicus) breed of Brahman (Brahman) With the gene amplification and restriction fragment length polymorphism method (PCR-RFLP) existing RAPD (Random Amplified Polymorphic DNA) or SCAR (Sequence Characterized Amplified Regions) in order to distinguish it from the Otherwise, more accurate and reproducible discrimination is possible. Applying the PCR-RFLP technique using the SRY gene, it can be usefully used to determine the foreign introduced beef derived from Hanwoo beef and Boss Indicus.

Hereinafter, the present invention will be described in more detail with reference to Examples. The technical spirit of the present invention is not limited by the following examples.

EXAMPLE

1. Materials

159 cows (156 cows of the 32th generation, 156 cows of Korean cattle, 3 cows of Chungbuk National University) and the outpatient introduction of cattle ( Bos indicus , Brahman variety) 23 genomic DNAs were extracted and the DNA concentration was constant to 50 ng / μl and used for PCR reaction.

2. Design and Synthesis of SRY Primer

Based on the bovine sequence registered in the GenBank ( Bos Taurus) taurus ) strain (AB039748) and SRY genes from bovine indices-derived foreign cows were examined and their structural differences were compared.

The SRY gene according to this embodiment is a gene of Bos taurus variety, and is a single nucleotide polymorphism (SNP) including a portion in which the 1707 th T base is mutated to the G base.

The SRY gene is a primer for amplifying the 801bp fragment of the 1003 to 1803 nucleotide sequence of Bos taurus (AB039748) and the 273 to 1073 nucleotide sequence of Bos indicus (AY079145). Forward primers and reverse primers were designed and synthesized to have the nucleotide sequences shown in Table 1 below.

3. PCR amplification of the SRY gene

Reaction conditions for PCR amplification of the SRY gene are gene amplifiers (product name: GeneAmp PCR System 2700) was used under the following conditions. The reaction solution was placed in a PCR tube with 50ng of template DNA, 10x PCR buffer, 1.5 μl of MgCl ₂ , 0.5 μl of dNTP (10 mM), 10 pmol / μl of primer each, and Taq DNA polymerase (Ampli Taq). Gold, USA) 1 unit was added so that the PCR reaction solution was 25 μl total.

The PCR cycle was repeated for a total of 38 cycles of preheating at 94 ° C for 10 minutes, followed by 94 cycles of 30 seconds at 60 ° C, 30 seconds at 60 ° C, and 45 seconds at 72 ° C. Exited.

4. DNA cleavage and electrophoresis by restriction enzyme treatment

Restriction enzyme treatment of the 801bp PCR amplification product of the SRY gene generated after PCR was performed as follows. 15 μl of the amplification product was digested with Mse I (10 u / μl) restriction enzyme using a total volume of 20 μl and overnight at 37 ° C. DNA fragments obtained by cleavage with restriction enzymes (see Table 2) were electrophoresed with 3% agarose gel, isolated, and stained with Et-Br (ethidium bromide) solution. Bands were detected.

As shown in Table 2, the 1707th T base was changed to G base in Bos taurus , and two bands with sizes of 581 bp and 220 bp were detected, and Bos indicus was 581 bp, 123 bp and 97 bp. Three bands with were detected.

Referring to FIG. 4, it can be clearly seen whether the band is generated.

According to the method for determining the structural difference of the RFLP genotype of the SRY gene according to the present invention, Bos Taurus derived ( Bos from taurus ) and beef indigo Introduce Brahman accurately.

Although described with reference to the embodiments above, those skilled in the art will understand that the present invention can be variously modified and changed without departing from the spirit and scope of the invention as set forth in the claims below. Could be.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> METHOD OF IDENTIFICATION BETWEEN HANWOO AND FOREIGN BEEF MEATS <130> APC-05-0121 <140> 10-2005-0127393 <141> 2005-12-21 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> SRY-F <400> 1 ttagaacgct tacaccgcat attacttc 28 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> SRY-R <400> 2 tagtaaaatt gagataaaga gcgcctttgt tag 33  

Claims (7)

DNA extracted from Hanwoo (Bos taurus) and foreign imported cattle, and then sex-determining region Y (SRY) gene (AB039748) of Bos taurus varieties registered in the Genbank. PCR using a forward primer of 5'-TTAGAACGCTTACACCGCATATTACTTC-3 '(SEQ ID NO: 1) and a reverse primer of 5'-TAGTAAAATTGAGATAAAGAGCGCCTTTGTTAG-3' (SEQ ID NO: 2) to include the position where the 1707 th T base is G-based Amplification and digestion of the PCR amplified amplification product with restriction enzyme Mse I followed by analysis of single nucleotide polymorphism (SNP) to distinguish between genotypes of Hanwoo and foreign-imported cattle. How to determine the right. The method of claim 1, wherein the foreign introduced cattle are Brahman species derived from Boss indicus. delete delete delete The method of claim 1, wherein in the amplification step, 801bp fragments of the 1003 to 1803 nucleotide sequence of Bos taurus DNA (registration number: AB039748) and the 273 to 1073 nucleotide sequence region of the boss indicus DNA (registration number: AY079145) The amplification method characterized in that the amplified Hanwoo and foreign introduced cattle. The method of claim 1, wherein the Korean cattle have two polymorphisms and the foreign introduced cattle have three bands.

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