Single Molecule Genotyping Using Microfluidic Stagnation Point Flows

描述（由申请人提供）：目前用于确定染色体DNA上的单核苷酸多态性（SNP）的方法需要微阵列（Affyellow，Inc. - http://www.affymetrix.com/index.affx;Perlegen Sciences-http：//www.perlegen.com/）或单分子梳理技术结合光学基因作图（OpGen，Inc. --http://www.opgen.com）。这些方法通常在给定的测试中区分单个染色体上的可能几百个SNP，并且不产生超过1 Kbp的真实单倍型信息。此外，DNA测序和微阵列技术受到DNA杂交过程缓慢的限制。我们建议使用在流动装置中分离和拉伸单个大DNA分子的最新进展，特别是停滞点流动，以开发基于与结合到荧光珠的探针的序列特异性杂交的快速单分子基因组测序（SNP和标签SNP）技术。这种新的微流体过程有可能通过在几次扫描中揭示数千个SNP并将处理时间缩短到几个小时（每千个SNP）来彻底改变SNP基因分型。正如HapMap项目所揭示的那样，人类基因组中有多达30万个标签SNP。因此，我们提出的主要问题是：是否有可能开发一种单分子方法，通过利用DNA的微流体操纵来完成全基因组扫描？为了解决这个问题，我们需要了解的耦合之间的流体力学和DNA分子的动力学，杂交反应的动力学，和分子生物学技术与杂交探针的发展。我们建议通过开发这些方法来演示实现这一目标的技术？噬菌体DNA，多联体？- 噬菌体DNA、T4以及E.大肠杆菌DNA，后者包括超过1 Mbp的基因组长度。我们建议开发的技术来完成一个完整的标签SNP扫描的生物体的基因组，包括人类基因组，使用单分子技术在微流体。人类基因组的完整标签SNP扫描，在几天内廉价完成，为个性化医疗时代奠定了基础。用于医学的全基因组关联研究，包括疾病的遗传倾向的早期预测以及对药物反应的基因组易感性，可以变得广泛使用，从而对药物基因组学领域产生巨大影响。

DESCRIPTION (provided by applicant): Current methods for determining Single Nucleotide Polymorphisms (SNPs) on chromosomal DNA require either microarrays (Affymetrix, Inc. - http://www.affymetrix.com/index.affx; Perlegen Sciences -- http://www.perlegen.com/) or single molecule combing techniques combined with optical gene mapping (OpGen, Inc. -- http://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.