Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen

AlphaScreen technology allows the development of high-throughput homogeneou s proximity assays. In these assays, signal is generated when 680 nm laser fight irradiates a donor bead in close proximity to an acceptor bead. For t he detection of nucleic acids, donor and acceptor beads are brought into pr oximity by two bridging probes that hybridize simultaneously to a common ta rget and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stran ded DNA target. The combination of AlphaScreen with allele-specific amplifi cation (ASA) and allele-specific hybridization (ASH) has allowed the develo pment of two homogenous single-nucleotide polymorphism (SNP) genotyping pla tforms. Both types of assay are very robust, routinely giving accurate geno typing results with <2 ng of genomic DNA per genotype. An AlphaScreen valid ation study was performed for 12 SNPs by using ASA assays and seven SNPs by using ASH assays. More than 580 samples were genotyped with accuracy >99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats wit h volumes as small as 2 mul, allowing a considerable reduction in the amoun t of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-through put genotyping.