SNP genotyping of unpurified PCR products by sandwich-type affinity electrophoresis on a microchip with programmed autonomous solution filling.

We demonstrate rapid single-nucleotide polymorphism (SNP) genotyping on a poly(dimethylsiloxane)-glass microchip. Sandwich-type affinity electrophoresis was employed to achieve sufficient specificity for reliable genotyping of unpurified PCR products. We tested three SNPs in different genes: CYP2D6 of artificial templates, and ALDH3A1 and CYP1A1 of human genomic samples. The target sequences were amplified by asymmetric PCR. For each SNP, we prepared a capture probe-poly(dimethylacrylamide) (CP-PDMA) conjugate and allele-specific, fluorescently-labeled detection probes (DPs). Prior to the electrophoresis, necessary solutions--the amplified sample, the CP-PDMA conjugate, the DPs, and a washer--were autonomously filled into their own regions of the microchannel in contact with each other. For precise control of this filling process, we have extended our published technique to a "programmed" version, in which additional passive stop valves synchronized the solution contacting events. Then we electrophoretically carried out a target DNA hybridization step, a DP hybridization step, and a washing step at the CP-PDMA conjugate region. This 3-step electrophoresis was completed in 2 min. The formation of the sandwich hybridization complex (CP-target-DP) was evaluated by fluorescence. Normalized fluorescence values of the different genotypes were clearly and reproducibly discriminated. The assay format presented here will be suitable for SNP genotyping at the point of care.