Development of Tetra Primer Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS - PCR) for the Detection of Thyroglobulin (TG5) SNP in Cross Breed Wagyu

Abstract

Amplification refractory mutation system PCR (ARMS–PCR) was designed to detect single nucleotide polymorphism (SNP) in cross breed Wagyu thyroglobulin (TG5) gene. The C422T SNP has been shown to be related to marbling phenotype. Other detection methods including PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing have high sensitivity. However, it is relatively time-consuming, and high cost compared to ARMS-PCR. Therefore ARMS-PCR technique was developed to reduce time and cost for SNPs detection. Genomic DNA extracted from follicles hair was used as templates for PCR amplification. Three different genotypes were observed. The samples with PCR products of 400 bp and 199 bp indicated homozygous (T/T), those with 400 bp and 275 bp products indicated homozygous (C/C) and the ones with three products of 400 bp, 275 bp and 199 bp were heterozygous (C/T). The SNP detection by ARMS-PCR was compared with the conventional method of PCR-RELP. The results were found to be consistent. This work showed that the ARMS-PCR can be successfully applied to the detection of TG5 C422T SNP in cattle. The ARMS-PCR method is more economical, easy to perform, and precise genotyping can be observed. Results from this study suggest that ARMS-PCR procedure is a useful technique for SNPs genotyping. ARMS-PCR to detect SNPs of other meat quality associated gene will be further developed.