Integrated allele-specific polymerase chain reaction–capillary electrophoresis microdevice for single nucleotide polymorphism genotyping

Abstract

An integrated allele-specific (AS) polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for multiplex single nucleotide polymorphism (SNP) genotyping on a portable instrumentation, which was applied for on-site identification of HANWOO (Korean indigenous beef cattle). Twelve sets of primers were designed for targeting beef cattle's eleven SNP loci for HANWOO verification and one primer set for a positive PCR control, and the success rate for identification of HANWOO was demonstrated statistically. The AS PCR and CE separation for multiplex SNP typing was carried out on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for microvalve function. The operation of the sample loading, AS PCR, microvalve, and CE on a chip was automated with a portable genetic analyzer, and the laser-induced fluorescence detection was performed on a miniaturized fluorescence detector. The blind samples were correctly identified as a HANWOO by showing one or two amplicon peaks in the electropherogram, while the imported beef cattle revealed more than five peaks. Our genetic analysis platform provides rapid, accurate, and on-site multiplex SNP typing.