KR100780449B1 - New primer set and method for discriminating Korean beef - Google Patents

Abstract

The present invention relates to a novel primer set and a method for discriminating Hanwoo beef using the same, and more particularly, to a novel primer set and PCR performed using the same to detect a unique insertion-deletion site of Hanwoo (Indel, Insertion and Deletion) As a method of discriminating Korean beef from imported beef by analyzing the results, it is possible to quickly and easily discriminate Korean beef from various kinds of imported meat, which can be effectively used in the distribution and retrieval process of Korean beef.

Indel, Insertion and Deletion site, primer set, Korean beef

Description

New primer set and method for discriminating Korean beef meat using same {A NEW PRIMER SET AND METHOD OF SELECTING HANWOOMEAT BY USING THE SAID PRIMER SET}

1 is a Bos according to an embodiment of the present invention. Hanwoo BAC terminal nucleotide sequence is distinguished from the genomic data of Tarus (AAFC01456374),

Figure 2 shows a Hanwoo BAC terminal nucleotide sequence distinguished from genomic data of Bos Tarus (AAFC01616858), according to an embodiment of the present invention,

Figure 3 is an electrophoresis picture of the PCR results for the samples of Hanwoo, dairy cows and foreign cattle carried out using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, according to an embodiment of the present invention,

Figure 4 is an electrophoretic picture of the PCR results for Hanwoo, dairy cows and foreign cattle samples carried out using the primers of SEQ ID NO: 3 and SEQ ID NO: 4, according to an embodiment of the present invention to be.

[Industrial use]

The present invention relates to a new primer set and, in detail, to a new primer set and a method for discriminating Korean beef using the same, which are designed to search for a unique insertion-deletion site of Hanwoo.

[Private Technology]

In recent years, the consumption of meat has increased as the Korean economy has been rapidly westernized along with the growth of the national economy and the increase of income. Beef demand has also increased. Meanwhile, as the beef market is fully opened, imported beef is indiscriminately entering the domestic market, and imported beef, which is relatively cheaper than Korean beef, is quickly dominating the domestic beef market.

Domestic livestock farmers' strategy to increase the demand for beef and open the beef market is to raise high quality Korean beef meat to differentiate it from imported meat. Due to the high quality of Hanwoo beef, the price gap between Hanwoo beef and imported beef has deepened. Due to this price gap, there are frequent cases of distributional irregularities in which cheap imported beef is sold as expensive Hanwoo beef. For example, there are several cases related to the sale of vulnerable products, such as the sale of mixed beef and imported meat into Hanwoo beef gift sets at department stores and large-scale discount stores.

The sale of bluffs has been pointed out as another cause of weakening the competitiveness of Hanwoo beef. In order to eradicate this, the meat transaction record duty system was established and the government continued to crack down, but there is a limit to cracking down on the sale of the pack.

The limitation of the crackdown is caused by the lack of scientific and objectively secured beef and imported meat discrimination techniques. In other words, the development of the technology for discriminating pure beef cattle, beef and imported beef is essential to block the sale of bovine meat and protect the beef cattle farmers and beef market.

On the other hand, since the breeding of cows is generally a method of relying on the appearance, the shape, and the like, the identification of the breed of the cattle slaughtered and converted to meat form is virtually impossible by the conventional method. Therefore, in order to determine the breed of the slaughtered meat, that is, the breeding stock, immunoassay (ELISA) and isoelectric point electrophoresis (IEF) have been used. However, a problem has been raised that the above-mentioned discrimination method is almost impossible to use for discriminating among the varieties within the same species.

Recently, with the development of molecular biology and genetic analysis techniques, techniques for solving the problems of the above methods, that is, the identification of varieties in the same species using the difference of DNA sequences have been proposed. Most of the discriminating techniques are based on polymorphism of DNA, and specific examples thereof include Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP), Single Strand Conformational Polymorphism (SSCP), and facilitation of collapse. Discrimination techniques include Decay Accelerating Factor (DAF), Microsatellite, Random Amplified Polymorphic DNA (RAPD), and Sequence Characterized Amplified Regions (SCAR) primers that extend part of the nucleotide sequence of RAPD primers.

However, the above-described discrimination techniques also have problems as a practical method for discriminating Korean beef. As a specific example, among the above-described identification techniques, Korean beef cattle discrimination method (Byeong-rok Min, Lee Mu-ha, Han Jae-yong, Beef varieties (Korean beef, beefstein (Holstein), imported beef) using RAPD), Korean Society for Animal Science Vol. 37 (6) ): 651-660 (1995)) has a problem in that it is difficult to put to practical use because of the low reproducibility and accuracy of the results due to the sensitivity of PCR amplification conditions.

In addition, Hanwoo discrimination method using Hanwoo specific SCAR primer that partially extended the base sequence of RAPD primer (Hong Young Ho, Jung Il Jung, Tae Heon Kim, Heebal Kim, Doo Hak Kim, Hyung Sun Cho, Byeong Wook Han, Jae Yong Yong, Hanwoo discrimination marker using breed specificity) Development, Vol. 2 (2): 107- 114 (1998), was found to be relatively stable compared to the RAPD method, but this problem was also pointed out in the reliability of discrimination.

Recently, discrimination techniques using the MC1R (Melanocortin 1 Receptor) gene, which is known to be involved in the dark and red color expression, have been proposed.

Specific examples include the method of discriminating Hanwoo cattle using PCR-RFLP using three restriction enzymes such as Bse118Ⅰ, MspⅠ and Aci I (Kim Tae-heon, Yoon Doo-hak, Park Eung-woo, Lee Hye-young, Oh Sung-jong, Jung Il-jeong, Tak Tae-young, Kim Kyung-nam, Han Jae-yong, and Cattle Breeds by Melanocortin Receptor 1 A Study on Genotype Frequency of (MC1R) Gene, Journal of Animal Science and Technology Vol 42 (6): 735-744 (2000)) and PCR-RFLP Methods Using Two Restriction Enzymes, BsrFⅠ and MspⅡ (Eui-Ryong Jung, Woo-Tae Kim, Yeon-Soo Kim, Han Sang, Hanwoo beef discrimination using the PCR-RFLP marker of the bovine groping-related gene MC1R, Animal Resource Journal Vol 42: 379-390 (2000)).

The method of discriminating Hanwoo beef using the MC1R gene is very efficient and economical in distinguishing Hanwoo beef from cow meat (Holstein species) and some imported meat (black Angus species). There is a problem in that it is not possible to distinguish between the limousine species having a similar color to Korean beef, the simanthal species having a light yellowish brown color to the deep reddish brown color, or the hybrids of Hanwoo and cows (Holstein).

The present invention solves the problems of the existing method of discriminating Korean beef and imported beef as described above, to provide a novel primer set and a method of discriminating Korean beef using the same for quickly and accurately discriminating various kinds of imported beef and Korean beef The purpose.

In order to achieve the above object, the present invention

As a primer for Korean priority selection selected from the group consisting of polynucleotides having a size of 20bp to 50bp consecutively among polynucleotides consisting of the first base to the 50th base on the 5 'end of the nucleotide sequence shown in SEQ ID NO:

The 5 'end of the primer is the 5'-terminal first base of the nucleotide sequence shown in SEQ ID NO: 5,

The primer 3 'end is a primer of any one of the 20th to 50th base sequence of the base sequence shown in SEQ ID NO: 5 and

As a primer for Korean priority selection selected from the group consisting of polynucleotides having a size of 20 bp to 50 bp consecutively among polynucleotides consisting of 1 st base to 50 th base of the 3 'end of the nucleotide sequence shown in SEQ ID NO:

The 5 'end of the primer is any one of the bases 200 to 229 of the base sequence shown in SEQ ID NO: 5,

The primer 3 'end provides a set of primers for screening Hanwoo, consisting of a primer that is the first base of the 3'-end side of the nucleotide sequence shown in SEQ ID NO: 5.

In addition, the present invention

As a primer for Korean priority selection selected from the group consisting of polynucleotides having a size of 20bp to 50bp consecutively among polynucleotides consisting of the first base to the 50th base on the 5 'end of the nucleotide sequence shown in SEQ ID NO:

The 5 'end of the primer is the 5'-terminal first base of the nucleotide sequence shown in SEQ ID NO: 6,

The primer 3 'end is a primer of any one of the 20th to 50th base sequence of the nucleotide sequence shown in SEQ ID NO: 6 and

As a primer for Korean priority selection selected from the group consisting of polynucleotides having a size of 20bp to 50bp consecutively among polynucleotides consisting of 1st to 50th bases on the 3 'end of the nucleotide sequence shown in SEQ ID NO:

The 5 'end of the primer is the base of any one of the 234th to 263th base sequence of the base sequence shown in SEQ ID NO: 6,

The primer 3 'end provides a set of primers for screening Hanwoo, consisting of a primer that is the first base of the 3'-end side of the nucleotide sequence shown in SEQ ID NO: 6.

The primer set for Hanwoo selection is preferably a primer set consisting of a primer set consisting of a nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2 or a primer consisting of a nucleotide sequence of SEQ ID NO: 3 and a nucleotide sequence of SEQ ID NO: 4 Can be.

In addition, the present invention has a nucleotide sequence of SEQ ID NO: 1 at the 5 'end of the nucleotide sequence, a nucleotide sequence of SEQ ID NO: 2 at the 3' end of the nucleotide sequence, and a total number of bases 249 bp for the selection of polynucleotides or complement thereof Provided is a polynucleotide for screening Hanwoo having an intact sequence.

In addition, the present invention has a nucleotide sequence of SEQ ID NO: 3 at the 5 'end of the nucleotide sequence, has a nucleotide sequence of SEQ ID NO: 4 at the 3' end of the nucleotide sequence, the total number of bases 283 bp polynucleotides for screening Hanwoo or its complementary Provided is a polynucleotide for screening Hanwoo having a sequence.

Said Hanwoo selection polynucleotide is preferably selected for Hanwoo having a nucleotide sequence of SEQ ID NO: 5 or a complementary nucleotide sequence thereof or a nucleotide sequence of SEQ ID NO: 6 or a complementary nucleotide sequence thereof For polynucleotides.

In another aspect, the present invention provides a Hanwoo screening polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 7 or a complementary sequence thereof, or a Hanwoo screening polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 8 or a complementary sequence thereof.

In addition, the present invention

a) collecting a DNA sample from beef that is to be discriminated; And

b) amplifying the sample DNA using a primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; And

c) by analyzing the amplification products, to provide a method for discriminating Korean beef cattle, comprising the step of identifying the Hanwoo specific polynucleotide fragments.

In step c), the amplification products may be electrophoresed to identify DNA bands, and in the case of a single band, may be discriminated by Korean beef.

In addition, the present invention

DNA samples collected from beef to be discriminated;

At least one set of primers for screening Hanwoo selected from the group consisting of a set of primers for screening Hanwoo and a set of primers for screening Hanwoo consisting of SEQ ID NO: 3 and SEQ ID NO: 2;

Amplification means capable of amplifying the DNA sample; And

Provided is a Korean beef cattle discrimination kit comprising a detection means for identifying Korean cattle specific polynucleotide fragments.

The Hanwoo specific polynucleotide fragment may be a nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence of SEQ ID NO: 6, wherein the Hanwoo specific polynucleotide fragment is 69 of the entire sequence of the polynucleotide of SEQ ID NO: 5 which is a native marker unique to Hanwoo Adenine (Adenine, A), which is the 77-degree base of the Hanwoo native indel-site or the entire polynucleotide of SEQ ID NO: 6 between thymine (T) and the 70th guanine (G) ) And a Hanwoo specific polynucleotide fragment capable of detecting the indel site unique to Hanwoo existing between the 78th base, Adenine (A).

In addition, the detection means after the electrophoresis of the product of the PCR reaction, it may be a means that can determine whether the DNA band resulting from the electrophoresis is a single band.

The present inventors compared and analyzed the DNA sequence of a cow and the DNA sequence of a foreign cow, and prepared a primer set capable of identifying and searching for an indel region unique to the cattle. The present invention has been completed by developing a method for discriminating Korean beef and foreign beef.

Hereinafter, the present invention will be described in detail.

The present invention provides a primer set for selecting Korean cattle.

Specifically, the present invention is a new primer set designed to search for indels (Indel, Insertion and Deletion) unique to foreign cows, that is, a cow that can search for a uniquely-inserted portion of Hanwoo. A set of primers for selection is provided.

In an embodiment of the present invention, the primer set for selecting Hanwoo may be composed of a forward primer of the 5 'end of the nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer of the 3' end represented by SEQ ID NO: 2.

In addition, the primer set for Hanwoo selection may be composed of a forward primer of the base 5 'terminal represented by SEQ ID NO: 3 and a reverse primer of the 3' terminal represented by SEQ ID NO: 4.

The sequence of the primers constituting the primer set for the selection of Hanwoo is as shown in Table 1 below.

division Sequence SEQ ID NO: Annealing temperature Set-1 Forward 5'-CCCTGTTTCACTCACCCATC-3 ' One 51 ℃ Reverse 3'-TATTTGGTTTTCCCCCGAAG-5 ' 2 Set-2 Forward 5'-TGATCAAGGTCACAGGAAGG-3 ' 3 62 ℃ Reverse 3'-TGAAGCTGGGATCAAAGAGC-5 ' 4

In the present invention, Hanwoo (Hanwoo) is a unique prop species of Korea for thousands of years to adapt to the Korean climate for a suitable constitution and refers to livestock that are reared (in one) or for breeding for meat.

The present invention also provides a polynucleotide molecule for screening Hanwoo.

In detail, the polynucleotide molecule may mean that the primer set for selecting Hanwoo and the native insertion-deletion site of Hanwoo are used.

In an embodiment of the present invention, the polynucleotide molecule has a forward primer represented by SEQ ID NO: 1 at the 5 'end of the nucleotide sequence, has a reverse primer represented by SEQ ID NO: 2 at the 3' end of the nucleotide sequence, and the total number of bases Polynucleotide or nucleotide sequence of 249 bp, characterized by the presence of a unique insertion-deletion site of Hanwoo between Thymine (T), the 69th base of the total sequence, and Guanine (G), the 70th base. It has a forward primer represented by SEQ ID NO: 3 at the end, and has a reverse primer represented by SEQ ID NO: 4 at the end of SEQ ID NO: 3, and has a total number of bases of 282 bp and a 77th base in the total sequence (Adenine, A ) And a 78-base adenine may be a polynucleotide characterized by the presence of an indel region unique to Hanwoo.

Preferably, the polynucleotide is a Hanwoo screening polynucleotide having a nucleotide sequence of SEQ ID NO: 5 or a complementary nucleotide sequence thereof, or a Hanwoo screening polynucleotide having a nucleotide sequence of SEQ ID NO: 6 or a complementary nucleotide sequence thereof Can be.

In addition, the polynucleotide for screening Hanwoo of the present invention may be a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 7 or a complementary sequence thereof or a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 8 or a complementary sequence thereof.

The native-deletion site unique to Hanwoo may be a deletion site that is predominantly specific to Hanwoo or Hanwoo as a result of comparison and search with genome data of foreign cows. In an embodiment of the present invention, the foreign genome data may be genomic data of Bos Tarus published in NCBI.

In an embodiment of the present invention, the insertion-deletion region of SEQ ID NO: 5 is composed of thymine (T), which is the 69th base, and guanine (G), which is the 70th base, that is, with foreign cases. Otherwise, no other base sequence is inserted between thymine (T), the 69th base, and guanine (G), the 70th base, and at least one base sequence is compared with a foreign base. It means fruitful. In addition, the insertion-deletion region of SEQ ID NO: 6 has adenine (Adenine, A), which is the 77th base, and adenine (Adenine, A), which is the 78th base, that is, unlike foreign cases, the entire sequence of SEQ ID NO: 6 It means that one or more nucleotide sequences are deleted in comparison with the base of a foreign base without another base sequence inserted between adenine (Adenine, A), which is the 77th base of the sequence, and adenine (Adenine, A), which is the 78th base.

In addition, in an embodiment of the present invention, the polynucleotide is a nucleotide sequence of SEQ ID NO: 7 or a complementary thereof including the polynucleotide represented by SEQ ID NO: 5, characterized in that there is a unique insertion-deletion site of Hanwoo It may be a Hanwoo screening polynucleotide having a sequence.

 In an embodiment of the present invention, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 8 or a complementary sequence thereof, including the polynucleotide represented by SEQ ID NO: 6, characterized in that a unique insertion-deletion site exists for Hanwoo It may be a polynucleotide for screening Hanwoo.

In addition, the present invention provides a method of discriminating Korean beef from Korean beef and imported meat using the Hanwoo screening primer set.

In another aspect, the present invention provides a Korean beef cattle determination kit (KIT) that can distinguish between Korean beef and imported meat using the primer set.

The discrimination method of Hanwoo beef that distinguishes the Hanwoo beef and imported beef is

a) collecting a DNA sample from beef that is to be discriminated; And

b) amplifying the sample DNA using a primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; And

c) by analyzing the amplification product, it may be a method of discriminating Korean beef meat, comprising the step of identifying a Korean cattle specific polynucleotide fragment.

In the present invention, the native marker of Hanwoo means a characteristic site of the Hanwoo gene which is distinguished from the importing cow, and in the embodiment of the present invention, it means an Indel, Insertion and Deletion site. . The Hanwoo native gene marker may be preferably an insertion-deletion (Indel, Insertion and Deletion) site of SEQ ID NO: 5 or an insertion-deletion (Indel, Insertion and Deletion) site of SEQ ID NO: 6.

In the present invention, the DNA sample taken from beef may be genomic DNA obtained from cow's blood, semen or gravy or beef tissue, preferably genomic DNA obtained from cow's gravy or beef tissue. .

The detecting of the native markers of the native cattle may be performed by a conventional gene sequence detection method. Preferably, the PCR sample is performed using the DNA sample as a template and the primer set for the selection of the cattle as a primer. In addition, after the electrophoresis of the product of the PCR reaction using an agarose gel (agarose gel) and a polyacrylamide gel (polyacrylamide gel), it is carried out by a method of confirming the DNA band (DNA band) resulting from the electrophoresis can do.

In a specific embodiment of the present invention, in the case of a sample taken from Korean cattle, the DNA band resulting from the electrophoresis appears as a single band, and in the case of a sample taken from an importing place, the DNA band resulting from the electrophoresis has a plurality of bands. It was confirmed that appears, and by confirming the number of the DNA bands based on this, if the DNA band of a particular beef is a single band can be determined as a han beef.

The PCR reaction can be carried out in the usual reaction conditions, preferably in the reaction conditions described in Table 2 according to the type of each primer set.

Primer set Step Temperature Reaction time YUHW-MBL-1 First (1 ^st ) denaturation 94 ℃ 3 minutes Second (2 ^nd ) denaturation 94 ℃ 30 seconds Annealing 51 ℃ 30 seconds Extension 72 ℃ 45 seconds Cycle go to 2 ^nd denaturation 35 cycle Final extension 72 ℃ 5 minutes YUHW-MBL-2 First (1 ^st ) denaturation 94 ℃ 3 minutes Second (2 ^nd ) denaturation 94 ℃ 30 seconds Annealing 62 ℃ 30 seconds Extension 72 ℃ 45 seconds Cycle go to 2 ^nd denaturation 35 cycle Final extension 72 ℃ 5 minutes

The beef cattle discrimination kit may include: a DNA sample collected from beef beef to be discriminated;

At least one set of primers for screening Hanwoo selected from the group consisting of a set of primers for screening Hanwoo and a set of primers for screening Hanwoo consisting of SEQ ID NO: 3 and SEQ ID NO: 2;

Amplification means capable of amplifying the DNA sample; And

Hanwoo beef determination kit comprising a detection means for identifying the Hanwoo specific polynucleotide fragments,

The Korean beef cattle discrimination kit is composed of a DNA sample collected from beef to be discriminated from a crowd consisting of a set of primers for screening Hanwoo, consisting of SEQ ID NO: 1 and SEQ ID NO: 2 and a set of primers for screening Hanwoo, consisting of SEQ ID NO: 3 and SEQ ID NO: 4 It may be composed of one or more selected Hanwoo cattle selection primer set and a detection means for detecting the gene marker unique to the Hanwoo.

The detection means may be a means for detecting a conventional gene marker, preferably may include a means for detecting the gene marker by performing a PCR reaction and electrophoresis. In an embodiment of the present invention, the detection means is a means for performing a PCR reaction using the primer set for the selection of Korean cattle and a means for confirming the number of DNA bands by performing electrophoresis on the product of the PCR It may be to include a means for detecting a gene marker having a.

Hereinafter, an Example demonstrates this invention in detail.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

<Example>

Example 1 Searching for Genetic Markers Specific to Korean Cattle

Example 1-1 Preparation of BAC Library (Bacterial Arificial Chromosome library, BAC library)

Among the 26th to 35th Hanwoo Protest Groups conducted by the National Agricultural Cooperative Federation for Hanwoo, the blood of Hanwoo was judged to be excellent in meat and meat as a result of slaughter. The collected blood was refrigerated at 4 ° C. with the addition of EDTA to prevent coagulation until used for chromosomal DNA extraction.

The fabrication of the BAC library was made by previously published methods (Cai L., Taylor JF, Wing RA, Gallagher DS, Woo SS, Davis SK Construction and Characterization of a Bovine Bacterial Artificial Chromosome Library.Genomics. 29, 413-425 (1995) and Osoegawa K., Woon PY, Zhao B., Frengen E., Tateno M., Catanese JJ, de Jong PJ An improved approach for construction of bacterial artificial chromosome libraries.Genomics. 52, 1-8 (1998) ).

Chromosomal DNA of leukocyte cells was isolated and purified from the collected blood using a DNA separation kit (Genotein, South Korea) and a protocol provided by the DNA separation kit manufacturer.

After the isolated and purified chromosomal DNA is prepared with an agarose plug, an existing method using the method (An Improved Approach for Construction of Bacterial Artificial Chromosome Libraries, Genomics 52: 1-8 (1998), BAC (Bacterial Artificial Chromosome) clones containing a total of 150,000 different regions of Hanwoo chromosome were prepared with reference to the present invention).

More specifically, the isolated and purified chromosomal DNA was digested using EcoR I and Hind III restriction enzymes (NEB, USA), and the cut Hanwoo chromosome DNA was digested with pBACe3.6 vector (Accession No. U80929) and pIndigoBAC-5. Each was ligated to a vector (Epicentre, WI, USA). The ligation was carried out in 15 μl 10 X T4 ligase buffer (ligase buffer, Fermentas, Hanover, USA), 3 μl T4 ligase (ligase, Fermentas, Hanover, USA) in 50ng of pBACe3.6 vector or pIndigoBAC-5 vector. 300 ng of inserted DNA, ie, the digested Hanwoo chromosome DNA, was mixed and the total amount was adjusted to 150 μl with ultra high purity water at 16 ° C. for overnight reaction.

The recombinant vector produced by the ligation is transformed into a bacterial competent cell (DH-10B, Invitrogen, USA) using an electrophorator, and then plated onto an LB plate. Only BAC clones containing chromosomal DNA of Hanwoo on LB plates were selected and inoculated in 96 deep well plates containing 1 ml of 2 X LB medium and incubated for 20 hours. Glycerol stock was prepared by mixing 40 μl of the culture solution and 10 μl of glycerol, and then stored in a deep freezer at -70 ° C.

Example 1-2 Isolation and Sequencing of Hanwoo BAC DNA

Isolation of BAC DNA was performed by performing both terminal sequencing on 10,272 clones of the 1500,000 clones prepared as described below.

First, the BAC clone of Hanwoo prepared above was inoculated into a 2.0 ml 96 deep well plate (Bioneer, South Korea) containing 1.5 ml of 2 X LB medium containing 12.5 μg / ml of chloramphenicol, followed by shaking. Using an incubator (shaking incubator, MegaGrow (Bioneer, South Korea), shaking culture for 18 hours at 450rpm.

The cultured shake cultures were extracted using Montage BAC ₉₆ miniprep kit (Millpore), alkaline lysis (Birnaboim , HC and Doly, JA A rapid alkaline extraction procedure for screening recombinant plasmid DNA.Nucleic Acids Res . 7 (6): 1513-1523 (1979) and Kelley, JM, Fields, CE, Craven, MB, Bocskai, D., Kim, U., Rounsley, SD and Adams, MD 1999.High throughput direct end sequencing of BAC clones Nucleic Acids Res . 27 (6): 1539-1546.) Was used to isolate plasmid, that is, Hanwoo BAC DNA. Electrophoresis was performed on the isolated Hanwoo BAC DNA to confirm the integrity and concentration of the DNA.

After confirming the integrity and concentration of the DNA, PCR was performed using a universal sequencing primer. More specifically, the universal sequencing primer used a T7 primer (5'-TAATACGACTCACTATAGGG-3 ') and an engineered M13R primer (modified M13R primer, 5'-GCGGATAACAATTTCACACAGG-3'). Was performed.

Step Temperature Reaction time First (1 ^st ) denaturation 95 ℃ 1 minutes Second (2 ^nd ) denaturation 95 ℃ 30 seconds Annealing 50 ℃ 10 seconds Extension 60 ℃ 45 seconds Cycle go to 2 ^nd denaturation 35 cycle

The DNA amplified by the PCR was subjected to capillary sequencing using an automated DNA sequencer, 3700 capillary sequencer and 3730 XL capillary sequencer (Applied Biosystems, USA), to sequence terminal sequences.

The base sequence of the BAC clone whose sequence was confirmed by the sequencing was registered in the gene bank of the National Center for Biotechnology Information (NCBI).

In order to increase the reliability of the nucleotide sequence of the BAC clone sequence identified by the sequencing, the nucleotide sequence of the vector used for constructing the clone was removed, and a clone having a phred score of 20 or more and a terminal nucleotide sequence of 100 base pairs or more was obtained. Screening was performed using the Phred program (Washington Univ). Repeated masking using a repeat masking program (Repeat Masker-open-3-1-2, 'cross_match version 0.990329') and Rebase Update (20051025), which is a repeat database, of the selected Hanwoo BAC clone That is, the removal of the repeat base sequence was performed.

Base sequence from which the repeat base sequence was removed and Bos published in NCBI The similarity of Tarus genomic data was investigated using BLAST (Basic Local Alignment Search Tool). The BLAST program used for the similarity investigation was used NCBI local BLAST, the database (NCBI Genome Bos Taurus (October 27, 2005)).

Example 1-3 Search for Genetic Markers Unique to Korean Cattle

Hanwoo's unique genetic marker, that is, the part showing the difference between the nucleotide sequence of foreign cow and foreign cow, is searched by comparing the genome data of Hanwoo, which has deleted the repeat base sequence, and the genome data of Bos Tarus published in NCBI. Indel, Insertion and Deletion sites were identified.

Based on the comparative search result, the part showing the difference between the base sequence of Korean cattle and foreign cows using the Primer 3 program (http: //frodo.wi.mit.deu/cgi-bin/primer3/primer3_www.cgi) -The sequence of the primer including the deletion (Indel, Insertion and Deletion) site was determined, and the Hanwoo BAC terminal nucleotide sequence including the sequence of the insertion-deletion site and the primer is shown in FIGS. 1 and 2.

In detail, the comparison result with genome data of Bos Tarus (genome data, AAFC01456374), which is foreign genome data disclosed in the NCBI, was found between thymine, the 69th base, and guanine, the 70th base. The presence of the deletion site was confirmed by the gene marker unique to Hanwoo, and the sequence of the primer capable of detecting the gene marker was determined by the above method and named YUHW-MBL-1.

A Hanwoo BAC terminal nucleotide sequence including the identified gene marker and primer sequence is shown in FIG. 1. The underlined portion of FIG. 1 shows the nucleotide sequence of the primer YUHW-MBL-1.

In addition, as a result of comparative search with Bos Tarus ' genome data (genome data, AAFC01616858), which is foreign genome data disclosed in the NCBI, among the nucleotide sequences shown in FIG. 2, that is, between the 149th base adenine and the 150th base adenine, It was confirmed that a deletion site exists between adenine, a 77th base of SEQ ID NO: 6, and adenine, a 78th base, and confirmed this by using a unique Korean marker. The sequence of a primer capable of detecting the gene marker was identified in the above method. Was determined and named YUHW-MBL-2.

A Hanwoo BAC terminal nucleotide sequence including the identified gene marker and primer sequence is shown in FIG. 2.

The underlined portion of FIG. 2 shows the nucleotide sequence of the primer YUHW-MBL-2.

Example 2 Preparation of a Primer That Can Detect Genetic Expression Unique to Hanwoo

Bioneer (Daejeon, South Korea), Inc. based on the determined sequences of the primers YUHW-MBL-1 and YUHW-MBL-2, in order to detect the Hanwoo native gene markers identified in Examples 1-3. The primer was prepared by requesting synthesis.

The primer produced by requesting the synthesis was as described in Table 1 above.

Example 3 Discrimination of Korean Cattle and Imports

Determination of Hanwoo and imported cows using the primers YUHW-MBL-1 and Primer YUHW-MBL-2 prepared in Example 2 is a sample taken from the material, namely already confirmed Hanwoo, dairy cow (Holstein species) and imported beef. Was carried out in the following manner.

Example 3-1: Collection of Samples

Example 3-1-1: Sampling of Korean Cattle and Dairy Cattle

Blood was collected from 275 Hanwoo cattle and 68 cows (Holstein species), which were judged by a veterinarian in Gyeongsang-do and Jeolla-do slaughterhouses, and EDTA (0.5 M, pH) was added to 50 mL of collected blood to prevent coagulation of the collected blood. 8.0) 10 mL was added to the falcon tube, and stored at -80 ° C until genomic DNA extraction.

Example 3-1-2: Sampling of Imports

In the case of imported cattle, 63 samples of imported beef were collected from department stores and large discount stores located in Daegu and Gyeongsan, and semen samples from 13 foreign breeds and 181 specimens were obtained. The collected beef sample and semen secured were stored at -80 ℃ until genomic DNA extraction.

Example 3-2 Extraction of Genomic DNA from Collected Samples

Example 3-2-1 Extraction of Genomic DNA from Blood Samples

Extraction of genomic DNA from the blood sample of Example 3-1-1 was performed using a FlexiGene DNA kit (FlexiGene DNA kit, Qiagen GmbH, Hilden, Germany) and a protocol supplied by the kit manufacturer.

Example 3-2-2 Extraction of Genomic DNA from Beef Samples

1 g of tissue isolated from the imported beef sample of Example 3-1-2 contained 5 to 10 mL of PBS buffer (PBS buffer, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). The mixture was put into a falcon tube and ground using a homogenizer, and then filtered using gauze. After centrifugation was performed at 300 x g for 5 minutes on the filtered sample solution, pellets were collected. Genomic DNA was extracted from the collected pellets using the same kit and protocol as in Example 3-2-1.

Example 3-2-3 Extraction of genomic DNA from semen samples

Take 30 μl of semen from the semen sample of the imported source of Example 3-1-2 and transfer it to a 1.5 ml tube, followed by 10 mM TNE buffer I, 10 mM Tris-HCl (pH 8.0). 500 μl of 10 mM EDTA, 1% SDS, 100 mM NaCl, 100 ug / mL proteinase K) was added. The TNE buffer I-added semen sample tube is mixed well using a vortex and then placed in a 70 ° water bath and incubated for 3 hours to hydrolysis. Was performed. The hydrolyzed mixture was centrifuged at 12,800 x g and 25 ° C using a centrifuge to collect pellets containing sperm.

0.5 ml of TNE buffer II (TNE buffer I + 0.1 M DTT) was added to the pellet, and then placed in a 55 ° C. water bath and incubated for 8 hours. 500 ul of reaction solution (Phenol / Chloroform / Isoamyl alcohol solution, Phenol, Chloroform and Isoamyl alcohol is mixed at 25: 24: 1) is added to the sample that has been incubated at room temperature for 30 minutes. Shaking was shaken. After centrifugation of the mixed solution (shaking) for 5 minutes at 12,000 rpm and room temperature (15 ℃) using a centrifuge, the supernatant was transferred to a new tube (tube), the same capacity of isopro Panol (isopropanol) was added. The mixed solution to which isopropanol was added was centrifuged at 13,000 xg and 4 ° C. for 15 minutes using a centrifuge. Then, isopropanol was removed, 1,000 μl of 70% ethanol was added, and 13,000 was added. DNA was washed by centrifugation for 5 minutes at xg conditions. The washed DNA was dried at room temperature (15 ° C.), and 50 μl of 1 × TE buffer (TE buffer, 10 mM Tris-Cl, 1 mM EDTA, pH 8.0) was added to the dried DNA pellet. After dissolving, genomic DNA was extracted by incubation at 4 ° C. for 16 hours. The genomic DNA was confirmed by performing electrophoresis on 1.2% agarose gel.

Example 3-3 PCR with Primer YUHW-MBL-1 and Primer YUHW-MBL-2

The reaction solution for PCR reaction was genomic DNA, Taq DNA polymerase extracted from 2 μl, 3 ng of 10 X reaction buffer (reaction buffer, 100 mM Tris-Cl, 400 mL KCl, 20 mM MgCl2, pH 9.0, GenDocs, South Korea). 0.5 unit (5 unit / μl, GenDocs, South Korea), 1 μl of dNTPs mixture (2.5 mM dNTPs) and 10 pmoles of primer YUHW-MBL-1 or primer YUHW-MBL-2 (forward primer and reverse primer) After that, sterile distilled water was added to make a final volume of 20 μl.

The PCR reaction solution prepared above was subjected to a PCR reaction using a MyGenie 96 Gradient thermal cycler (Bioneer, Korea) under the conditions described in Table 4.

Primer set Step Temperature Reaction time YUHH-MBL-1 First (1 ^st ) denaturation 94 ℃ 3 minutes Second (2 ^nd ) denaturation 94 ℃ 30 seconds Annealing 51 ℃ 30 seconds Extension 72 ℃ 45 seconds Cycle go to 2 ^nd denaturation 35 cycle Final extension 72 ℃ 5 minutes YUHH-MBL-2 First (1 ^st ) denaturation 94 ℃ 3 minutes Second (2 ^nd ) denaturation 94 ℃ 30 seconds Annealing 62 ℃ 30 seconds Extension 72 ℃ 45 seconds Cycle go to 2 ^nd denaturation 35 cycle Final extension 72 ℃ 5 minutes

Example 3-4: Determination of Korean Beef Meat by Electrophoresis

Electrophoresis was performed on the PCR product in order to determine the Hanwoo beef using the PCR product amplified by Example 3-3.

To perform electrophoresis, a 1.2% agarose gel was prepared using 1 X TAE buffer (1 X TAE buffer, 40 mM Tris-base, 1 mM EDTA, 20 mM Acetic acid) and agarose. After loading the PCR product, electrophoresis was performed at 100 volt for 50 minutes to confirm the reaction.

After confirming the PCR reaction through the presence or absence of the reaction, the PCR product was subjected to secondary electrophoresis using a non-denature polyacrylamide gel. The 8% non-denature polyacrylamide gel used in the secondary electrophoresis was 20% acrylamide-bis solution (40% acrylamide solution) and 2% Bis solution (Bio-rad, USA) Prepared by mixing 29: 1 ratio), 4.8 ml of sterile distilled water and 2.4 ml of 5 X TBE buffer (5 X TBE buffer, 45 mM Tris-base, 1 mM EDTA, 45 mM Boric acid) were mixed. . The gel was hardened by adding 200 ul of 10% Ammonium persulfate (Sigma, USA) and 10 ul of TEMED (Sigma, USA) to the prepared non-denature polyacrylamide gel.

5 μl of the PCR product of Example 3-3 was mixed with 1 μl of 6 × loading buffer, 0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol. After loading into each well of the polyacrylamide gel, electrophoresis was performed for 20 minutes at 85 volt and 1 hour 20 minutes using Mini Format Vertical Electrophoresis (Bio-rad, USA). .

The DNA size marker (DNA size marker) used to analyze the electrophoresis result was used a 100 bp DNA ladder (Bioneer, South Korea).

After completing the electrophoresis, 2 μl of EtBr (10 mg / ml) was stained and DNA bands were identified using ChemiImager ^™ Ready (Alpha Innotech, Mississauga, Ontario, Canada). 3 and FIG. 4.

The results shown in FIG. 3 are the results for 24 Hanwoo, 20 dairy cows and 24 foreign cows which were subjected to PCR using the primer YUHW-MBL-1, and the results shown in FIG. 4 using the primer YUHW-MBL-2. Results were obtained for each of 20 cattle, cows, and foreign cows.

As shown in FIG. 3, the number of individuals showing DNA bands of different sizes other than 249 base pairs was 0 for Korean cattle, 18 for foreign cattle, and 3 for dairy cows.

Table 5 shows the analysis results of the PCR products for all the Korean cattle, dairy cows, and foreign cattle collected in Example 3-1.

division Hanwoo milk cow Foreign cattle Multi-Band Confirmation Number 6/275 5/68 122/244 percentage(%) 2% 7% 50%

As shown in Table 5, only 6 out of 275 samples, or 2%, had double bands in Korean cattle, whereas 122 out of 244 samples, 50% had multiple bands in foreign cattle. In the case of cows, five of the 68 samples showed a plurality of bands different from the primer size.

Based on the above results, when PCR was performed using the primer YUHW-MBL-1, a sample showing a plurality of bands (ie, bands different from the primer size) is meat of a foreign beef, ie, imported meat, rather than Korean beef. The probability was confirmed to be high.

In addition, as shown in FIG. 4, the number of individuals showing DNA bands of different sizes other than 282 base pairs was 2 in Korean cattle, 12 in foreign cattle, and 4 in dairy cows.

Table 6 shows the analysis results of the PCR products for all the Korean cattle, dairy cows, and foreign cattle collected in Example 3-1.

division Hanwoo milk cow Foreign cattle Multi-Band Confirmation Number 37/275 12/68 108/244 percentage(%) 13% 18% 44%

As shown in Table 6, only 37 out of 275 samples, or 13%, had double bands in Korean cattle, whereas 108 out of 244 samples, 44% had multiple bands in foreign cattle. In the case of cows, 12 of the 68 samples showed a plurality of bands different from the primer size.

Based on the above results, when PCR was performed using the primer YUHW-MBL-2, a sample showing a plurality of bands (ie, bands different from the primer size) is meat of a foreign beef, that is, imported meat day, rather than Korean beef. The probability was confirmed to be high.

Referring to the results of the above experiment, using the indel, insertion and deletion (Indel, Deletion) region of the native cow's own genetic markers, to make a primer set that can detect this, using PCR and electrophoresis after using By analyzing the differences in the DNA band patterns of electrophoresis, the present invention distinguishes between Korean cattle and foreign cows, compared to conventional methods using the MC1R (Melanocortin 1 Receptor) gene, which is known to be involved in groping. In a short time, it is possible to distinguish between Korean beef and imported meat in a simple way.

As described above, when using the primer set for the selection of Korean beef, not only the imported meat of various varieties can be discriminated as compared to the conventional method, but also the Korean beef can be quickly and easily identified without the help of an expert. At any point in the process, it is possible to distinguish between Korean beef and imported meat, which is considered to be widely used industrially.

Attach sequence list electronic file  

Claims (16)

As a primer for Korean priority selection selected from the group consisting of polynucleotides having a size of 20bp to 50bp consecutively among polynucleotides consisting of the first base to the 50th base on the 5 'end of the nucleotide sequence shown in SEQ ID NO: The 5 'end of the primer is the 5'-terminal first base of the nucleotide sequence shown in SEQ ID NO: 5, The primer 3 'end is a primer of any one of the 20th to 50th base sequence of the base sequence shown in SEQ ID NO: 5 and As a primer for Korean priority selection selected from the group consisting of polynucleotides having a size of 20 bp to 50 bp consecutively among polynucleotides consisting of 1 st base to 50 th base of the 3 'end of the nucleotide sequence shown in SEQ ID NO: The 5 'end of the primer is any one of the bases 200 to 229 of the base sequence shown in SEQ ID NO: 5, The primer 3 'end is a primer set for screening Hanwoo consisting of a primer which is the first base of the 3'-end side of the nucleotide sequence shown in SEQ ID NO: 5 The method of claim 1, The primer set is a primer set for Hanwoo selection, which is a primer consisting of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2. As a primer for Korean priority selection selected from the group consisting of polynucleotides having a size of 20bp to 50bp consecutively among polynucleotides consisting of the first base to the 50th base on the 5 'end of the nucleotide sequence shown in SEQ ID NO: The 5 'end of the primer is the 5'-terminal first base of the nucleotide sequence shown in SEQ ID NO: 6, The primer 3 'end is a primer of any one of the 20th to 50th base sequence of the nucleotide sequence shown in SEQ ID NO: 6 and As a primer for Korean priority selection selected from the group consisting of polynucleotides having a size of 20bp to 50bp consecutively among polynucleotides consisting of 1st to 50th bases on the 3 'end of the nucleotide sequence shown in SEQ ID NO: The 5 'end of the primer is the base of any one of the 234th to 263th base sequence of the base sequence shown in SEQ ID NO: 6, The primer 3 'end is a primer set for screening Hanwoo consisting of a primer that is the first base of the 3'-end side of the nucleotide sequence shown in SEQ ID NO: 6 The method of claim 3, The primer set is a primer set for Hanwoo selection, which is a primer consisting of the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4. Hanwoo having a nucleotide sequence of SEQ ID NO: 1 at the 5 'end of the nucleotide sequence, a nucleotide sequence of SEQ ID NO: 2 at the nucleotide sequence 3', and having a polynucleotide or a complementary sequence thereof for selecting Hanwoo having a total number of 249 bp Screening Polynucleotides. The method according to claim 5, wherein the polynucleotide is Hanwoo screening polynucleotide having a nucleotide sequence of SEQ ID NO: 5 or a complementary nucleotide sequence thereof. Hanwoo having a nucleotide sequence of SEQ ID NO: 3 at the 5 'end of nucleotide sequence, a nucleotide sequence of SEQ ID NO: 4 at the nucleotide sequence of 3' end, and having a polynucleotide or a complementary sequence for Hanwoo selection having a total number of 283 bp Screening Polynucleotides. The method of claim 7, wherein the polynucleotide is Hanwoo screening polynucleotide having a nucleotide sequence of SEQ ID NO: 6 or a complementary nucleotide sequence thereof. A polynucleotide for screening Hanwoo consisting of the nucleotide sequence of SEQ ID NO: 7 or a complementary sequence thereof. A polynucleotide for screening Hanwoo consisting of the nucleotide sequence of SEQ ID NO: 8 or a complementary sequence thereof. a) collecting a DNA sample from beef that is to be discriminated; And b) amplifying the sample DNA using a primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; And c) analyzing the amplification product, and identifying a Korean beef specific polynucleotide fragment. The method of claim 11, Wherein c) step is to identify the DNA band by electrophoresis the amplification product, if the single band discrimination of Korean beef. DNA samples collected from beef to be discriminated; At least one set of primers for screening Hanwoo selected from the group consisting of a set of primers for screening Hanwoo and a set of primers for screening Hanwoo consisting of SEQ ID NO: 3 and SEQ ID NO: 2; Amplification means capable of amplifying the DNA sample; And Hanwoo beef determination kit comprising a detection means for identifying a Hanwoo specific polynucleotide fragment. The method according to claim 13, wherein the unique Hanwoo unique gene marker is unique to Hanwoo existing between thymine (T), the 69th base of the total sequence of the polynucleotide of SEQ ID NO: 5 and guanine (G), the 70th base Han Beef determination kit, characterized in that the insertion-deletion site. The method according to claim 13, wherein the unique Hanwoo native gene marker is unique to the Hanwoo existing between the adenine (Adenine, A) of the 77th base of the polynucleotide of SEQ ID NO: 6 and the adenine (Adenine) of the 78th base Han Beef determination kit, characterized in that the insertion-deletion site. The method according to any one of claims 13 to 15, wherein the detection means comprises a means for confirming whether or not the DNA band resulting from the electrophoresis is a single band after electrophoresis of the product of the PCR reaction. Han beef meat discrimination kit, characterized in that the means.

Images