Allele-specific polymerase chain reaction for the discrimination of elite Korean cattle associated with high beef quality and quantity

Abstract

Techniques such as direct sequencing and PCR-RFLP (restriction fragment length polymorphism) are widely used to analyze the genotypes of livestock. However, these conventional methods have the disadvantage of taking a lot of time and incurring considerable cost. The allele-specific PCR method performs PCR using two primers, and a single nucleotide polymorphism (SNP) genotype can be identified through electrophoresis, saving time and cost. Highly accurate results can be obtained by designing specific primers according to the allele of the SNP under study, utilizing primer binding to a complementary matching sequence. In this study, we established a genotyping system with the AS-PCR technique, using SNPs related to the improvement of the meat quality and meat mass of Korean cattle. Using the PRIMER1 program, we designed specific primers for SNPs located at the 3′ end, with one SNP marker in the HSPB1 gene related to meat quantity and two SNP markers in the ADH1C and FASN genes related to meat quality in cattle. AS-PCR was performed on 10 Korean cattle using the primers designed with this system, and the genotypes could be identified by the size of the PCR product amplified as a result of electrophoresis. In the case of the HSPB1 g.2352T > C SNP, the T allele was amplified to 148 bp, and the C allele was amplified to 222 bp. The ADH1C c.-64T > C SNP was amplified to 492 bp at the T allele and 330 bp at the C allele. The FASN g.17924G > A SNP A allele was amplified to 377 bp and the G allele to 507 bp. The results for each SNP genotype were verified using direct sequencing, which showed that the genotypes identified by direct sequencing and the genotypes identified by the AS-PCR method matched exactly. The AS-PCR method therefore appears to be valuable for use in a genotyping system.