FRET primer for single nucleotide polymorphism (SNP) genotyping

Choy Len Fong1, Fiona Ng1, Kanaga Sabapathy2, Kim Halpin1 1Life Technologies, Singapore; 2Laboratory of Molecular Carcinogenesis, National Cancer Centre, Singapore

Abstract
Single nucleotide polymorphisms (SNPs) have become one of the most important objects of medical research. They have the potential to identify new drug targets, explain individual differences in the effectiveness of drugs, and susceptibility to disease. However the identification of SNP profiles by high resolution melt (HRM) is currently limited. The goal of this study was to develop an accurate HRM assay which employs the use of fluorescence resonance energy transfer (FRET) primers for identifying SNPs. A FRET primer is internally labeled with a fluorescence dye at a thymine (T) nucleotide. A Fret primer assumes a randomly coiled structure as a single stranded primer, resulting in the reporter dye and quencher being in close proximity, hence quenching fluorescence. Upon Fret primer extension, a rigid duplex forms, resulting in reporter dye and quencher separation and hence increased fluorescence which is ideal for real-time PCR quantification and melt curve study. In this study, we describe the feasibility of using a Fret primer for genotyping SNPs with HRM-based genotyping, offering an alternative to an intercalating dye (i.e. SYTO-9)-based platform. A Fret primer targeting the p53 codon 72 SNP site was designed and tested for the ability to discriminate the two SNP homozygous populations by HRM on a real-time PCR platform. p53 is currently the most frequently mutated tumour-suppressor gene in human cancers. A SNP in codon 72 of p53 results in either an arginine (Arg) or proline (Pro) residue, and has been demonstrated to affect p53 function. Our results show that the FRET primer coupled with HRM analysis reliably enables discrimination between Arg/Pro SNP in 18 peripheral blood sample of cancer patients. The FRET primers exhibit comparable sensitivity and expansive dynamic range as compared to SYTO-9 based HRM platform.