Single Molecule Genotyping Using Microfluidic Stagnation Point Flows

描述(申请人提供)：目前确定染色体上单核苷酸多态(SNP)的方法需要微阵列(Affymetrix，Inc.-http://www.affymetrix.com/index.affx；PerLegen Sciences-http://www.perlegen.com/))或结合光学基因定位的单分子组合技术(OpGen，Inc.--http://www.opgen.com).一般来说，这些方法在给定的测试中可能区分单个染色体上的数百个SNP，并且不会产生超过1KBP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。我们建议利用流动装置中分离和拉伸单个大分子DNA分子的最新进展，特别是停滞点流动，开发一种快速的单分子基因组测序(SNP和Tag SNP)技术，该技术基于序列特异性杂交到结合到荧光微珠的探针。这种新的微流控技术有可能在几次扫描中发现数千个SNP，并将处理时间减少到几个小时(每1000个SNP)，从而有可能使SNP基因分型发生革命性变化。正如公开的HapMap项目所揭示的那样，人类基因组中有多达30万个标签SNPs。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完整的基因组扫描？为了解决这个问题，我们需要了解流体动力学和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。我们建议通过在噬菌体DNA、噬菌体DNA、T4和E.Coli DNA的串联体上发展这些方法来演示实现这一点的技术，后者的基因组长度超过1MBP。我们建议开发一种技术，利用微流体中的单分子技术，完成对包括人类基因组在内的生物体基因组的完整标签SNP扫描。对人类基因组进行完整的标签SNP扫描，以低廉的成本在几天内完成，为个性化医学时代奠定了基础。全基因组关联医学研究包括疾病遗传倾向的早期预测以及基因组对药物反应的易感性可以得到广泛的应用，从而对药物基因组学领域产生巨大的影响。

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.