Single Molecule Genotyping Using Microfluidic Stagnation Point Flows

描述（由申请人提供）：目前测定染色体DNA上单核苷酸多态性（snp）的方法需要微阵列（Affymetrix公司- http://www.affymetrix.com/index.affx； Perlegen科学公司- http://www.perlegen.com/）或单分子梳理技术结合光学基因定位（OpGen公司- http://www.opgen.com）。这些方法，一般来说，在一个给定的测试中，可能在单个染色体上区分几百个SNPs，并且不能产生超过1Kbp的真正的单倍型信息。此外，DNA测序和微阵列技术受到DNA杂交过程缓慢的限制。我们建议利用流动装置中单个大DNA分子的分离和拉伸的最新进展，特别是停滞点流动，开发一种基于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，以结合荧光珠的探针。通过在几次扫描中显示数千个SNP，并将处理时间缩短到几小时（每千个SNP），这种新的微流体工艺有可能彻底改变SNP基因分型。正如公共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的微流体操作来完成全基因组扫描？为了解决这个问题，我们需要理解水动力学和DNA分子动力学之间的耦合，杂交反应的动力学，以及与杂交探针开发相关的分子生物学技术。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， ？-噬菌体DNA， T4和大肠杆菌DNA，后者包括基因组长度超过1mbp。我们建议开发技术来完成一个完整的标签SNP扫描生物体的基因组，包括人类基因组，使用微流体中的单分子技术。对人类基因组进行完整的标签SNP扫描，成本低廉，在几天内完成，为个性化医疗时代奠定了基础。医学基因组全关联研究，包括疾病遗传倾向的早期预测以及对药物反应的基因组倾向，可以得到广泛应用，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.