DOE Human Genome Program Contractor-Grantee
29. Rapid and Accurate Detection of Human Functional SNPs Using a Base Stacking Microelectronic DNA Chip
Glen Evans1, David Canter2, Purita Ramos1, Ray Radtkey2, Ron Sosnowski2, Gene Tu2, James O'Connell2, and Michael Nerenberg2
1Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, TX and 2Department of Molecular Biology, Nanogen, Inc., San Diego, CA
Large scale genomic sequencing is revealing thousands of useful functional and non-functional single nucleotide polymorphisms (SNPs). Technology for the accurate, rapid and expandable assessment of large numbers of human SNPs in parallel is needed for research and medical applications. We describe a novel technology for SNP assessment on microelectronic silicon-based DNA chips that utilizes short fluorescently-labeled oligonucleotide reporters and targets of amplified source DNA. This assay takes advantage of base stacking energies in the design of probes and has the ability of accommodate different sized amplicons in parallel. This assay has been utilized to assay functional SNPs in parallel controlle by electronic fields induced on the chip surface and two-color fluorescence detection. A panel of model functional SNPs has been developed to evaluate the utilize of this method. These markers include polymorphic sites in genes for HH (hemochromatosis), Factor V, EH1 (epoxide hydrolase 1), EPHX2 (epoxide hydrolase 2) CYP19 (cytochrome P450), DTD (diastrophic dysplasia sulfate transporter), GSTA1 (alpha glutathione S transferase), GSTA12 (microsomal glutathione S transferase), NAT1 (N-acetyl transferase 1), NAT2 (N-acetyl transferase 2), ColA1 (type IV collagen), ApoCIII (apolipoprotein cIII), MGC24 (PNA-binding glycoprotein), PPP2R1B (lung cancer susceptibility polymorphism) and others. SNP detection from amplified genomic target DNA can be carried out on 100 SNPs on a single microelectronic chip with accuracy exceeding that of DNA sequencing. We have utilized this system for the systematic genotyping of more than 200 individuals for 15 SNPs, also determined by DNA sequencing, with virtually 100% accuracy. This system is imminently suited to point-of-care genetic diagnosis, forensic applications, medical diagnostics as well as large scale human genotyping for pharmacogenomics applications.
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