A microfluidic chip for rapid single nucleotide polymorphism (SNP) genotyping using primer extension on microbeads

Abstract

A microfluidic device, which performs a primer extension technique onto microbeads, is demonstrated to genotype Single Nucleotide Polymorphism (SNP) on genomic DNA. The device has on-chip heaters and sensors to create a uniform and yet stable temperature gradient between 56°C and 94°C across an array of serpentine microchannels, where the microbeads carry allele-specific primers passing through for thermal cycling. As the primers on the beads is designed to be only extended when perfectly matched with the SNP site of the template, the mismatched samples will not be amplified. The SNP discrimination is achieved. In addition that the microfluidic environment promote enhanced mass transport and better hybridization kinetics, the microbeads offer an advantage for faster thermal response and higher signal-to-noise ratio. Three most-frequent variants in one SNP location from genomic DNA of Taiwan country chicken are successfully distinguished in only five thermal cycles, compared to thirty (30) thermal cycles in traditional approaches. Moreover, the SNP genotyping procedures, which typically involves multiple steps in amplification and detection, were integrated onto a single chip.