Multiplex allele specific PCR 방법을 이용한 한우고기와 젖소고기의 신속한 판별

Rapid differentiation of Hanwoo and Holstein meat using multiplex allele specific polymerase chain reaction protocols

  • Koh, Ba-Ra-Da (Gwangju Metropolitan Health & Environment Research Institute)
  • 심사 : 2005.08.22
  • 발행 : 2005.09.22


Here I describe a multiplex allele specific PCR-based approach for the rapid detection between Hanwoo and Holstein meat associated with Melanocortin 1 receptor (MC1R) gene. Specific and universal oligonucleotide primers were used in combination to detect the presence of a single nucleotide polymorphism within the bovine MC1R DNA sequence. The presence of the bovine MC1R gene is indicated by the production of a single control PCR product, whilst positive samples generate an alternative smaller specific product over the same region. The mutations in MC1R104 codon revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. As little as 0.39 ng and 1.56 ng of genomic DNA of Hanwoo and Holstein could be detected by MAS-PCR assay, respectively. This technique, which is widely used in human genetic screening, provides a reliable and sensitive result that has not been documented for the identification of bovine coat color. The MAS-PCR assay approach was proven to be useful in complementing routine beef DNA analysis for differentiation of these MC1R variants and it would facilitate the screening of deceiving sales of Holstein meat in the butcher shop.


  1. 김태중, 박성도, 이재일. 소 모색관련 MC1R 유전자 의 SNP와 관련한 3'-tailed primer를 이용한 한우육의 판별. 한국동물자원과학회지 2004, 46, 897-902
  2. 김태헌, 윤두학, 박응우, 이혜영, 오성종, 정일정, 탁태영, 김경남, 한재용. 소 품종별 Melanocortin Receptor 1 (MC1R) 유전자의 유전자형 빈도에 관한 연구. 한국동물자원과학회지 2000, 42, 735-744
  3. 민병록, 한재용, 이무하. RAPD 기법을 이용한 쇠고기의 품종(한우육, 유우육 (Holstein육), 수입우육) 구분. 한국동물자원과학회지 1995, 37, 651-660
  4. 정의룡, 김우태, 김연수, 한상기. 소 모색관련 유전자 MC1R의 PCR-RFLP Marker를 이용한 한우육 판별. 한국동물자원과학회지 2000, 42, 379-390
  5. 정의룡, 김우태, 김연수, 한상기. PCR- SSCP 기법을 이용한 소 MC1R 유전자의 다형성 분석 및 한우육 감별. 한국동물자원과학회지 2001, 43, 45-52
  6. Abravaya K, Carrino JJ, Muldoon S, Lee HH. Detection of point mutations with a modified ligase chain reaction (Gap-LCR). Nucleic Acids Res 1995, 23, 675-82
  7. Ayyadevara S, Thaden JJ, Shmookler Reis RJ. Discrimination of primer 3'-nucleotide mismatch by taq DNA polymerase during polymerase chain reaction. Anal Biochem 2000, 284, 11-18
  8. Berryere TG, Schmutz SM, Schimpf RJ, Cowan CM, Potter J. TYRP1 is associated with dun coat colour in Dexter cattle or how now brown cow? Anim Genet 2003, 34, 169-175
  9. Brookes AJ. The essence of SNPs. Gene 1999, 234, 177-186
  10. Cone RD, Lu D, Koppula S, Vage DI, Klungland K, Boston B, Chen W, Orth DN, Pouton C, Kesterson RA. The Melanocortin Receptors : Agonists, Antagonists, and the Hormonal Control of Pigmentation. Recent Prog Horm Res 1996, 51, 287-317
  11. Ferrie RM, Schwarz MJ, Robertson NH, Vaudin S, Super M, Malone G, Little S. Development, multiplexing, and application of ARMS tests for common mutation in the CFTR gene. Am J Hum Genet 1992, 51, 251-262
  12. Kijas JMH, Wales R, Tomsten A, Chardon P, Moller M, Anderssson L. Melanocortin receptor 1 (MC1R) mutations and coat color in pigs. Genetics 1998, 150, 1177-1185
  13. Klungland H, Vage DI, Gomez-Raya L, Adalsteinsson S, Lein S. The role of melanocyte-stimulating hormone (MSH) receptor in bovine coat color determination. Mamm Genome 1995, 6, 636-639
  14. Kriegesmann B, Dierkes B, Leeb T, Jansen S, Brenig B. Two breed-specific bovine MC1-R alleles in Brown swiss and saler Breeds. J Dairy Sci 2001, 84, 1768-1771
  15. Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ. Effects of primer-template mismatches on the polmerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 1990, 18, 999-1005
  16. Livak KJ. Allelic discrimination using fluorogenic probes and the 5' nuclease assay. Genet Anal 1999, 14, 143-149
  17. Marklund L Johansson M, Sandberg K, Andersson L. A missense mutation in the gene for melanocytestimulating hormone receptor (MC1R) is associated with the chesnut coat color in horses. Mamm Genome 1996, 7, 895-899
  18. McPeake SR. Color pattern inherritance in beef cattle. University of Arkansas Division of Agriculture Cooperative Extension Service, Little Rock, 2004
  19. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988, 16, 1215
  20. Mokrousov I, Otten T, Filipenko M, Vyazovaya A, Chrapov E, Limeschenko E, Steklova L, Vyshnevskiy B, Narvskaya O. Detection of isoniazid-resistant Mycobacterium tuberculosis strains by a multiplex allele-specific PCR assay targeting katG codon 315 variation. J Clin Microbiol 2002, 40, 2509-2512
  21. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989, 17, 2503-2516
  22. Riley PA. Melanogenesis and melanoma. Pigment Cell Res 2003, 16, 548-552
  23. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified betaglobin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 1986, 324, 163-166
  24. Schork NJ, Fallin D, Lanchbury S. Single nucleotide polymorphisms and the future of genetic epidemiology. Clin Genet 2001, 58, 250-264
  25. Takeuchi S, Takahashi S. Melanocortin receptor genes in the chicken-tissue distributions. Gen Comp Endocrinol 1998, 112, 220-231
  26. Vage DI, Klungland H, Lu D, Cone RD. Molecular and pharmacological characterization of dominant black coat color in sheep. Mamm Genome 1999, 10, 39-43