Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres

Abstract

Here we report proof‐of‐principle for a microsphere‐based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure‐specific cleavage reaction that achieves single base discrimination with a 5′‐nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid‐support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.