Current methods for determining Single Nucleotide Polymorphisms (SNPs) on chromosomal DNA require either microarrays (Affymetrix, Inc. - www.affymetrix.com/index.affx; Perlegen Sciences -- www.perlegen.com/) or single molecule combing techniques combined with optical gene mapping (OpGen, Inc. -- www.opgen.com). These methods, in general, distinguish perhaps a few hundred SNPs on a single chromosome in a given test and do not produce true haplotype information over more than 1Kbp. Moreover, DNA sequencing and microarray technologies are limited by the slow process of DNA hybridization. We propose to use recent advances in the isolation and stretch of single, large DNA molecules in flow devices, particularly stagnation point flows, to develop a rapid, single molecule, genomic sequencing (SNP and tag SNP) technology based on sequence- specific hybridization to probes bound to fluorescent beads. This new microfluidic process has the potential to revolutionize SNP genotyping by revealing thousands of SNPs in a few scans and reducing processing times to a few hours (per thousand SNPs). As the public HapMap project reveals, there are as many as 300K tag SNPs in the human genome. Thus, the primary question that we pose: Is it possible to develop a single molecule method to complete a full genomic scan by exploiting microfluidic manipulation of the DNA? To address this question, we require an understanding of the coupling between hydrodynamics and the dynamics of DNA molecules, the kinetics of hybridization reactions, and the molecular biology techniques associated with hybridization probe development. We propose to demonstrate the techniques to accomplish this by developing these methods on ?-phage DNA, concatemers of ? - phage DNA, T4 as well as E. Coli DNA with the latter including genomic lengths in excess of 1 Mbp. We propose to develop the techniques to accomplish a complete tag SNP scan of an organism's genome, including the human genome, using single molecule techniques in microfluidics. A complete tag SNP scan of the human genome, accomplished inexpensively and in a few days, sets the stage for the era of personalized medicine. Genome wide association studies for medicine including early prediction of genetic tendencies for disease as well as genomic predispositions to drug response can become widely used, with the resulting enormous impact on the field of pharmacogenomics. ? ? ?
