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Nucleic Acids Research, 2003, Vol. 31, No. 11 e66
© 2003 Oxford University Press

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

Kakuturu V. N. Rao, Priscilla Wilkins Stevens, Jeff G. Hall1, Victor Lyamichev1, Bruce P. Neri1 and David M. Kelso

Department of Biomedical Engineering, Robert R. McCormick School of Engineering and Applied Science, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3107, USA and 1 Third Wave Technologies, 502 South Rosa Road, Madison, WI 53719-1256, USA

*To whom correspondence should be addressed. Tel: +1 847 491 7671; Fax: +1 847 491 4928; Email: rajarao{at}northwestern.edu

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.


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