SNP Analysis by Allele-Specific Extension of Fluorescently Labeled Nucleotides in a Microfluidic Flow-Through Device

Abstract

This report describes a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single nucleotide polymorphism (SNP). The method takes advantage on the fact that the reaction kinetic differs between matched and mismatched configurations of allele specific primers hybridized to DNA template. By combining reaction kinetics of the extension reaction and flow kinetics of a microfluidic device, mismatched configurations were discriminated from matched configurations. All three possible variants of a SNP site at codon 72 of the p53 gene were scored using our approach. The result in this work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of filter-chambers for high throughput SNP analysis.