An integrated allele-specific polymerase chain reaction-microarray chip for multiplex single nucleotide polymorphism typing.

An integrated allele-specific polymerase chain reaction (AS PCR) and microarray chip has been developed for multiplex single nucleotide polymorphism (SNP) typing on a portable genetic analyzer instrumentation. We applied the integrated PCR-microarray system for on-site Hanwoo (Korean indigenous beef cattle) identification. Eleven sets of primers were designed, among which ten sets of primers targeted ten SNP loci to discriminate Hanwoo from the imported beef cattle and one primer set was used as a positive PCR control. The AS PCR for multiplex SNP typing was conducted 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 micropump and microvalve function. The resultant AS PCR products were mixed with a hybridization buffer in a micromixer channel through the micropumping operation, and then the microarray assay was performed in the downstream process. Eleven duplicate probes were spotted in a glass slide, which was connected at the end of the micromixer channel unit. When the mixed solution was injected into the disposable microarray chip, pneumatically actuated micropumping was executed to speed up the hybridization process by inducing the convective flow. The fluorescence signals on each spot were monitored by a miniaturized fluorescence scanner, and the Hanwoo was verified by detecting the number of fluorescent spots with three or fewer among eleven. An integrated portable PCR-microarray genetic analysis microsystem was first demonstrated for rapid, accurate, and on-site multiplex SNP typing to differentiate animal species.