Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres.
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.
Rao
Kakuturu V N
KV
Department of Biomedical Engineering, Robert R. McCormick School of Engineering and Applied Science, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3107, USA.
Stevens
Priscilla Wilkins
PW
Hall
Jeff G
JG
Lyamichev
Victor
V
Neri
Bruce P
BP
Kelso
David M
DM
D001057:Apolipoproteins E / Q000235:genetics; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D008863:Microspheres; D009841:Oligonucleotides / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
D001057:Apolipoproteins E; D005456:Fluorescent Dyes; D009841:Oligonucleotides