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（Affymetrix, Inc. - http://www.affymetrix.com/index.affx；Perlegen Sciences - http://www.perlegen.com/）或与光学基因作图相结合的单分子组合技术（OpGen, Inc. - http://www.opgen.com）。一般来说，这些方法在给定测试中可能会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单个大 DNA 分子分离和拉伸（特别是停滞点流动）方面的最新进展，开发基于与荧光珠结合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。这种新的微流体工艺有可能彻底改变 SNP 基因分型，通过几次扫描即可揭示数千个 SNP，并将处理时间缩短至几个小时（每千个 SNP）。正如公共 HapMap 项目揭示的那样，人类基因组中有多达 30 万个标签 SNP。因此，我们提出的首要问题是：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 α-噬菌体 DNA（α 的串联体）上开发这些方法来演示实现这一目标的技术。 - 噬菌体 DNA、T4 以及大肠杆菌 DNA，后者的基因组长度超过 1 Mbp。我们建议开发技术，利用微流体中的单分子技术完成生物体基因组（包括人类基因组）的完整标签 SNP 扫描。对人类基因组进行完整的标签 SNP 扫描只需花费几天的成本即可完成，为个性化医疗时代奠定了基础。医学领域的全基因组关联研究，包括疾病遗传倾向的早期预测以及药物反应的基因组易感性，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.