KR100871145B1 - Discrimination method of identification between cows and the primer used thereto - Google Patents

Abstract

The present invention is the Y nucleotide of the ADRB2 gene contained in SEQ ID NO: 1, the R nucleotide of the CSN3 gene contained in SEQ ID NO: 2, the R nucleotide of the MITF gene contained in SEQ ID NO: 3, the R nucleotide of the TFEB gene contained in SEQ ID NO: 4 Any one of the SNPs, and using the information obtained from the SNP provides a method of determining the prop species to determine a specific breed.

SNP, Korean Beef

Description

Discrimination method of identification between cows and the primer used only}

1 is a nucleotide sequence including ADRB2,

2 is a nucleotide sequence including CSN 3,

3 is a nucleotide sequence including a MITF,

4 is a nucleotide sequence including TFEB,

Figure 5 is a PCR-RFLP experiment results for each gene (top left: ADRB2, top right: CSN3, bottom left: MITF, bottom right: TFEB).

The present invention relates to a method for judging prop species, and more specifically, it is possible to scientifically determine beef that is not visually identifiable by searching for mutations in a gene that is specifically fixed for each prop species in the commercial breed of beef. The present invention relates to a method for judging a new prop species.

Research reports on the development of technology to distinguish bovine breeds by SNP DNA analysis have been reported by several domestic researchers (Hong et al., 1998; development of marker markers for discriminating Korean cattle using breed specificity). Cho et al., 1997, Use of DNA Markers by RAPD for Characterization of Cattle Breeds, Journal of the Korean Society for Animal Genetics, 1: 49-57. Lee et al. (1994, Discrimination of Korean Cattle by Nucleic Acid Analysis, Korean Journal of Animal Science, 36: 369-373) disclose a technique for discriminating Korean cattle and cows using RAPD analysis, but the average reliability is 95% or less. The SCAR marker reported by Hong et al. (1998) was evaluated for higher reliability and reproducibility than previous methods, but also did not achieve the desired accuracy.

The present invention has been proposed to solve the problems of the prior art as described above, the object of the present invention is to search for mutations in the genes specifically fixed for each prop species to determine the scientifically unidentifiable beef It is to provide a method for judging small species that can eradicate illegal distribution by developing DNA markers.

In order to achieve the above object, the present invention provides the Y nucleotides of ADRB2 (Bos taurus adrenergic, beta-2, receptor, surface) gene included in SEQ ID NO: 1, R of CSN3 (kappa-casein) gene included in SEQ ID NO: 2 The nucleotide, R nucleotide of the microphthalmia-associated transcription factor (MITF) gene contained in SEQ ID NO: 3, R nucleotide of the transcription factor EB (TFEB) gene included in SEQ ID NO: 4 is referred to as SNP, and from the SNP Provides a method of judging prop species that determines specific varieties using information obtained.

Preferably the method of the present invention provides a method of determining the prop species to determine the Hanwoo and non-Hanwoo from the SNP information.

Preferably, the method of the present invention, in determining the prop species, processing a predetermined restriction enzyme that recognizes and restricts the nucleotide sequence including the selected SNP, and analyzing the resultant obtained Provide a method of judgment.

Preferably, the method of the present invention includes the step of designing a primer comprising the nucleotide of the selected SNP position as the 3 'terminal nucleotide in the determination of the prop species, and analyzing the amplified product using the designed primer. Provides a method of judging the prop species.

In another aspect, the present invention is the Y nucleotide of the ADRB2 gene contained in SEQ ID NO: 1, R nucleotide of the CSN3 gene contained in SEQ ID NO: 2, R nucleotide of the MITF gene contained in SEQ ID NO: 3, R of the TFEB gene contained in SEQ ID NO: 4 Provided is a primer for determining a prop species comprising any one of the nucleotides as the 3 'terminal nucleotide.

Hereinafter, the content of the present invention in more detail as follows.

The present invention provides a method for determining prop species, which is particularly useful for distinguishing between Hanwoo and Non-Hanwoo. The method for determining the prop species according to the present invention uses the SNP information existing between each breed by analyzing the nucleotide sequences of four genes owned by a cow. Specific methods for determining the prop type using the SNP information may vary. For example, the SNP information may be used in a method of PCR-RFLP for judging small species, and may also be used in a method for PCR analysis by prototyping a primer having a nucleotide at the 3 'end of a SNP position.

Four genes of ADRB2, CSN3, MITF, and TFEB are used as the gene according to the present invention, and specific nucleotide sequences thereof are included in SEQ ID NOs: 1 to 4. SEQ ID NOs: 1 to 4 are products amplified by PCR of each of these genes, and these base sequences contain primer sequences used for amplification.

The present invention provides a method for determining the specific breed of cattle by finding the SNP position present in the gene. Specifically, the Y nucleotide (C or T), SEQ ID No. 2 and the gene shown in FIG. 2 (/ under the ADRB2-containing gene; underlined primer sequence, / is the enzyme restriction position) Is the R nucleotide (A or G) of the CSN3 gene included in the enzyme restriction position, the R nucleotide (A of the MITF gene included in SEQ ID NO: 3 and the gene shown in FIG. 3 (underlined primer sequence, / is a restriction position). Or G), the R nucleotide (A or G) of the TFEB gene included in SEQ ID NO: 4 and the gene shown in FIG. 4 (underlined primer sequence, / is restriction position, red is SNP) corresponds to SNP position.

According to the method of PCR-RFLP using these SNP positions, it is possible to specifically recognize a specific base sequence including each selected SNP site, for example, a base sequence including 3 to 6 nucleotides including the SNP. By treating the enzyme, it is possible to determine the breed of the cow from the resulting product. Restriction enzymes may be used by selecting one or more types from specific recognition of specific nucleotide sequences, and are not required to be limited to specific types. Specific types of restriction enzymes that can be used are known in the art and are commercially available.

PCR amplification of the resultant treated with restriction enzymes showed that the gene expression frequency was different for each variety due to the presence of SNP. For example, if ADRB2 gene is used as a target gene and Taq I is selected as a restriction enzyme, a single amplification product can be obtained in Korean cattle, whereas two amplification products are obtained for most foreign breeds. This is due to the fact that alleles are fixed in Korean cattle, while foreign alleles have different alleles at SNP positions.

When the specific SNP of each gene used in the present invention is used in the propane species judgment process, it is difficult to discriminate 100% of Hanwoo from non-Korean cattle by using only one gene. Therefore, when judging by using two or more of each of the above genes, it is possible to ensure the accuracy with a higher probability than when discriminating varieties, and the SNP information held by these individual genes is combined with other SNPs that can be found in the future. It is still effective because it can be used in the decision making process.

In addition to the PCR-RFLP method, in the present invention, primers designed as 3 'terminal nucleotides of the nucleotide of the SNP position using the SNP information may be used. Such primers may be designed with reference to the nucleotide sequence of any one gene selected from SEQ ID NOs: 1 to 4. Primers may typically consist of 10 to 30 bases, such that the nucleotide of the SNP position is located at the 3 'end, so as to include an appropriate number of bases in the gene sequence upstream containing the SNP Can be designed. Thus, when PCR amplification of genes of different alleles into a template using primers having such sequences, the amplification products cannot be obtained for any one, whereas alleles having fixed sequences are amplified. This makes it possible to have a significant difference in the amount of DNA amplification. Using this property, the above-described SNP information according to the present invention can be used to determine a specific variety by simply measuring the amount of gene amplification.

Hereinafter, the configuration and effects of the invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples.

Example 1 Confirmation of SNP

Primers described in Table 1 were prepared for four bovine genes, ADRB2, MITF, CSN3, and TFEB. PCR reaction buffer (500 mM KCl; 100 mM Tris-Cl, pH8.2; 15 mM MgCl ₂ ; 0.1% Triton X-100, TaKaRa Co, Japan) 2.5 μl, 2.5 μm dNTPs 2 μl, 10 pmole 1 μl of primer, 25-50 ng of genomic DNA and 1 U of Taq DNA polymerase (TaKaRa Co, Japan) were added and filled with sterile distilled water so that the final volume was 25 μl. PCR amplification reaction was carried out under the same conditions as in Table 2. After PCR was completed, 5 μl of the PCR product was amplified on 1.2% agarose gel.

In order to determine the presence of nucleotide variation in the amplified PCR product, the base sequences of 8 kinds of Korean beef and foreign beef were determined. In order to determine the nucleotide sequence, the PCR product was purified using a PCR purification kit (CoreBio Systems, Korea), the DNA concentration was measured, and the sequencing reaction was performed. The sequencing reaction was filled with 30ng of PCR product, 1uL of Bigdye terminator (Applied Biosystems, USA), 2uL of 5 × sequencing buffer, and 1.6pmol of primer, and filled with 10uL of sterile distilled water. Amplification reaction was carried out for 35 times at 60 ° C. for 4 minutes. After amplification was completed, purified with isopropanol and the sequence was determined by an automatic DNA sequencer ABI3730XL instrument. After completion of the sequencing, a single nucleotide polymorphism (SNP) was detected in the amplified product using a multiple nucleotide sequence analysis program. As shown in FIGS. 1 to 4, one characteristic SNP was detected from four genes. Could.

Example 2 Determination Test of Prop Species

For each SNP of four genes, PCR-RFLP method was used to investigate the gene frequencies of 8 varieties of Korean and foreign beef. Restriction enzymes used to analyze the SNP of each gene were TaqI (ADRB2), BsrI (MITF), MspI (CSN3), HaeII (TFEB). To determine the genotype of PCR products with restriction enzymes, 10 µl of the PCR amplified product was treated for 2 hours in an incubator at 37 ° C with 3U restriction enzyme, followed by 5 volt / cm in agarose gel with EtBr. After electrophoresis, photographs were taken and genotypes were determined as shown in FIG. 5 (top left: ADRB2, top right: CSN3, bottom left: MITF, bottom right: TFEB; lane 1: DNA marker, lane) 2: AA, lane 3: AA, lane 4: AB, lane 5: AB, lane 6: BB, lane 7: BB).

The gene frequencies of the SNPs of each gene and the 8 varieties of Korean beef and foreign beef were as shown in Tables 3, 4, 5 and 6.

Table 1 Primer Sequence for Each Gene

Gene name Primer Name DNA sequencing Size of PCR Product ADRB2 ADRB2Fw: SEQ ID NO: 5  5'-CTTGCCCATTCAGATGCACT-3 ' 575 bp ADRB2Rv: SEQ ID NO: 6  5'-TGCTGGAGTAGCCATTCCCATA-3 '  CSN3 CSN3Fw: SEQ ID NO: 7  5'-TTAGGTCACCTGCCCAAATT-3 ' 341 bp 8CSN3Rv: SEQ ID NO: 8  5'-GTTGTCTTCTTTGATGTCTC-3 '  MITF MITFFw: SEQ ID NO: 9  5'-GGAACGGACGAATAGTCTAC-3 ' 206 bp MITFRv: SEQ ID NO: 10  5'-AAAAGGAAGACACGTACCAA-3 '  TFEB TFEBFw: SEQ ID NO: 11  5'-ACTTGTGTGTGAAGTAGCC-3 ' 483 bp TFEBRv: SEQ ID NO: 12  5'-GACCCCACATTCTAACTTG-3 '

<Table 2> PCR amplification conditions using the designed primer

Temperature (℃) time Repeat count first 94 5 minutes One Main amplification denaturalization 94 30 sec 35 Annealing ※ 30 sec extension 72 60 seconds Last 72 10 minutes One

※ Annealing Temperature ADRB2 (62 ℃), CSN3 (56 ℃), MITF (62 ℃), TFEB (62 ℃)

<Table 3> Gene Frequency by Varieties of ADRB2 Gene SNP

kind Analysis head Gene frequency (%) A B Sharore 12 12.50 87.50 Simmental 30 13.33 86.67 Angus 13 0.00 100.00 Hairford 20 0.00 100.00 Brahmin 19 2.63 97.37 limousine 40 37.50 62.50 Brown swiss 27 20.36 79.64 Santa One 50.00 50.00 Hanwoo 566 0.00 100.00 Sum 728 15.15 84.85

<Table 4> Gene frequencies by type of CSN3 gene SNP

kind Analysis head Gene frequency (%) A B Sharore 12 100.00 0.00 Simmental 30 100.00 0.00 Angus 13 100.00 0.00 Hairford 20 100.00 0.00 Brahmin 19 73.69 26.31 limousine 40 100.00 0.00 Brown swiss 27 100.00 0.00 Santa One 100.00 0.00 Hanwoo 566 100.00 0.00 Sum 728 97.08 2.92

<Table 5> Gene Frequency by Type of MITF Gene SNP

kind Analysis head Gene frequency (%) A B Sharore 12 95.84 4.16 Simmental 30 98.33 1.67 Angus 13 76.93 23.07 Hairford 20 47.50 52.50 Brahmin 19 97.37 2.63 limousine 40 95.00 5.00 Brown swiss 27 100.00 0.00 Santa One 100.00 0.00 Hanwoo 566 100.00 0.00 Sum 728 90.11 9.89

<Table 6> Gene Frequency (%) of Varieties of TFEB Gene SNP

kind Analysis head Gene frequency (%) A B Sharore 12 100.00 0.00 Simmental 30 100.00 0.00 Angus 13 100.00 0.00 Hairford 20 100.00 0.00 Brahmin 19 2.63 97.37 limousine 40 100.00 0.00 Brown swiss 27 100.00 0.00 Santa One 100.00 0.00 Hanwoo 566 100.00 0.00 Sum 728 89.18 10.82

As described above, the present invention can be seen that the present invention can be usefully used for the identification of small species, in particular, the determination of Hanwoo and Non-Hanwoo. It is expected to bring tremendous economic benefits not only to Korean cattle farmers but also to consumers, and to contribute to the establishment of a sound distribution order.

<110> Republic of Korea <120> Discrimination method of identification between cows and the          primer used <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 572 <212> DNA <213> bovine <220> <221> gene (222) (1) .. (572) <223> gene configuring ADRB2 <400> 1 cttacccatt cagatgcact ggtaccgggc cagccacaag gaagccatca actgctatgc 60 taaggaaacc tgctgtgact tcttcacgaa ccaaccctat gccattgcct cctccattgt 120 gtccttctac cttcccctgg tggtcatggt cttcgtctac tccagggtgt tccaggtggc 180 caaaaggcag ctccagaaga tygacaaatc tgagggccgc ttccatgccc aaaacgtcag 240 tcaagtggag caggatgggc ggagcggtct aggacaacgc aggacctcca agttctactt 300 gaaggaacac aaagccctca agactttagg cattatcatg ggcactttca ccctgtgctg 360 gctgcccttc ttcattgtca acattgtgca cgtgatcaag gataacctca tccgtaagga 420 aatatacatc cttctaaact ggttgggcta catcaactcc gctttcaatc cccttatcta 480 ctgccggagc ccagatttca ggattgcctt ccaggagctt ctctgcctgc gcaggtcttc 540 attgaaggcc tatgggaatg gctgctccag ca 572 <210> 2 <211> 341 <212> DNA <213> bovine <220> <221> gene (222) (1) .. (341) <223> gene comprising CSN 3 <400> 2 ttaggtcacc tgcccaaatt cttcaatggc aagttttgtc aaatactgtg cctgccaagt 60 cctgccaagc ccagccaact accatggcac gtcacccaca cccacattta tcatttatgg 120 ccattccacc aaagaaaaat caggataaaa cagaaatccc taccatcaat accattgcta 180 gtggtgagcc tacaagtaca cctaccaccg aagcagtaga gagcactgta gctactctag 240 aagattctcc rgaagttatt gagagcccac ctgagatcaa cacagtccaa gttacttcaa 300 ctgcagtcta aaaactctaa ggagacatca aagaagacaa c 341 <210> 3 <211> 206 <212> DNA <213> bovine <220> <221> gene (222) (1) .. (206) <223> gene configuring MITF <400> 3 ggaacggacg aatagtctac tgtttcttgt tggattgggg ccacctaaaa cgtggttatr 60 ctggaaatgc tagaatataa tcactatcag gtgagcttta ttcttatatt caaagtctct 120 gaaatgtaat tcataaattg agtgtttcac cttttcgtgt tattgtagta gacacacagt 180 tttcagttgg tacgtgtctt ccttwt 206 <210> 4 <211> 482 <212> DNA <213> bovine <220> <221> gene (222) (1) .. (482) <223> gene configuring TFEB <400> 4 acttgtgtgt gaagtagccc ctgcccggcc tcactcctcc tgtcggcccc cgcaccctgt 60 gtggacttag tgcccgcttg gcagcccgtg ggttggaagc cccccaccca ggggcagccc 120 tcttcatggg gctgggcrgg aagaggcctt cagcccggct cctcccagag gtgaaatcgt 180 gagggcaagg cgagggggcg ccgcagaggg gggacgggat gtgctggagt taagaagaca 240 tcttaccttc gagcctggtc cccccatcag tgaaggagga ctcattggaa ccaggggtcc 300 agaagttcct tcttggaggc atggactggc ccaggggcrc ccaggcttgc ctttctggca 360 ctccccccac ccacaggcgg gcccccccac cccgctttgg tgctaacagc tctcctccag 420 gtggtgtgag ggcgggggtg gccagaagag ggggtgctgg gttcaagtta gaatgtgggg 480 tc 482  

Claims (5)

In order to determine the prop species, the Y nucleotide of the ADRB2 gene included in SEQ ID NO: 1, the R nucleotide of the CSN3 gene included in SEQ ID NO: 2, the R nucleotide of the MITF gene included in SEQ ID NO: 3, and the TFEB contained in SEQ ID NO: 4 Any one or more of the R nucleotides of the gene is a monobasic polymorphism (SNP), PCR amplification for the monobasic polymorphism (SNP) from the bovine gene, and then the PCR product is restricted to recognize the base sequence containing the selected SNP Method of determining the prop species, including the process of processing the enzyme, and the result obtained by electrophoresis. The PCR product of claim 1, wherein the PCR product is prepared using any one of a primer set selected from SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and SEQ ID NO: 11 and SEQ ID NO: 12 Method for judging the prop species, characterized in that the PCR amplification. delete Any one of the Y nucleotide of the ADRB2 gene included in SEQ ID NO: 1, the R nucleotide of the CSN3 gene included in SEQ ID NO: 2, the R nucleotide of the MITF gene included in SEQ ID NO: 3, and the R nucleotide of the TFEB gene included in SEQ ID NO: 4 And a 3 'terminal nucleotide, and PCR amplification with a primer consisting of 10 to 30 nucleotides in the sequence list, and then measuring the amount of gene amplification of the PCR amplification product. Any one of the Y nucleotide of the ADRB2 gene included in SEQ ID NO: 1, the R nucleotide of the CSN3 gene included in SEQ ID NO: 2, the R nucleotide of the MITF gene included in SEQ ID NO: 3, and the R nucleotide of the TFEB gene included in SEQ ID NO: 4 A primer for determining the prop species comprising 10 to 30 nucleotides in the sequence listing, comprising 3 ′ terminal nucleotides.

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