A simple and rapid method for detection of single nucleotide variants using tailed primer and HRM analysis

• Published in: Journal of animal reproduction & biotechnology (Online) Vol. 38; no. 4; pp. 209 - 214
• Main Authors: Baek, Hyeonguk, Choi, Inchul
• Format: Journal Article
• Language: English
• Published: 한국동물생명공학회(구 한국수정란이식학회) 31.12.2023; 사단법인 한국동물생명공학회
• Subjects: 축산학; high-resolution melt; single nucleotide polymorphism; allele specific; genotype
• ISSN: 2671-4639; 2671-4663
• DOI: 10.12750/JARB.38.4.209

Background: Single nucleotide polymorphisms (SNPs) are widely used genetic markers with applications in human disease diagnostics, animal breeding, and evolutionary studies, but existing genotyping methods can be labor-intensive and costly. The aim of this study is to develop a simple and rapid method for identification of a single nucleotide change. Methods: A modified Polymerase Chain Reaction Amplification of Multiple Specific Alleles (PAMSA) and high resolution melt (HRM) analysis was performed to discriminate a bovine polymorphism in the NCAPG gene (rs109570900, 1326T > G). Results: The inclusion of tails in the primers enabled allele discrimination based on PCR product lengths, detected through agarose gel electrophoresis, successfully determining various genotypes, albeit with some time and labor intensity due to the use of relatively costly high-resolution agarose gels. Additionally, high-resolution melt (HRM) analysis with tailed primers effectively distinguished the GG genotype from the TT genotype in bovine muscle cell lines, offering a reliable way to distinguish SNP polymorphisms without the need for time-consuming AS-PCR. Conclusions: Our experiments demonstrated the importance of incorporating unique mismatched bases in the allele-specific primers to prevent cross-amplification by fragmented primers. This efficient and cost-effective method, as presented here, enables genotyping laboratories to analyze SNPs using standard real-time PCR.