The goal of this proposal is to develop a single step method for the parallel scoring of single nucleotide polymorphisms (SNPs) in a surface array format. The approach is based upon a recently developed invasive cleavage assay for SNP scoring referred to as the 'Invader"""""""" assay (Third Wave Technologies, Inc.) As it is a signal amplification technology rather than a target amplification technology, this assay is not subject to the contamination/carryover problems characteristic of PCR. It is a very simple, robust, and isothermal assay well-suited for high throughput analysis. In the proposed surface-based version of the procedure (the """"""""Surface Invader Assay""""""""), addition to the surface of target human genomic DNA containing a given SNP allele will result in specific cleavage of a corresponding surface-immobilized probe oligonucleotide containing a fluorophore-quencher dye pair. This cleavage will occur between the fluorophore and the quencher, leaving the unquenched fluorophore attached to the surface. The fluorescence intensity of this unquenched fluorophore will be substantially greater than that of the quenched fluorophore, and thus the region of the DNA array containing that probe oligonucleotide will exhibit increased fluorescence intensity in a specific target-dependent fashion. By employing an array of such probe oligonucleotides, one for each SNP allele to be typed, a single addition of target human genomic DNA to the surface, along with the other needed assay reagents (buffer and enzyme), followed by incubation, washing, and fluorescence imaging steps, will yield the genotypes of the target DNA analyzed for each SNP represented in the array.
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