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Here we report proof-of-principle for a microsphere- based genotyping assay that detects single nucle- otide 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 hybrid- ization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluores- cent 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.

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