Single Molecule Genotyping Using Microfluidic Stagnation Point Flows

使用微流体停滞点流进行单分子基因分型

基本信息

批准号：
7479859
负责人：
ERIC S SHAQFEH
金额：
$ 16.33万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-08 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7479859
关键词：
Acceleration Address Bacteriophages Binding Chromosome Mapping Chromosomes Comb animal structure Complex Concatenated DNA Coupled Coupling DNA DNA Probes DNA Sequence Data Development Device Designs Devices Diffusion Disease Escherichia coli Feedback Fluorescent Probes Genes Genetic Genome Genomics Genotype Haplotypes Hour Human Genome In Situ In Situ Hybridization Kinetics Length Literature Medicine Methods Microarray Analysis Microfluidic Microchips Microfluidics Microscope Microspheres Molecular Biology Techniques Molecular Conformation Motion Nanostructures Numbers Numeric Rating Scale Oligonucleotide Probes Optics Organism Peptide Nucleic Acids Pharmaceutical Preparations Pharmacogenomics Polymers Positioning Attribute Predisposition Process Quantum Dots Range Rate Reaction Reading Research SNP genotyping Sampling Scanning Science Single Nucleotide Polymorphism Staging Stretching Techniques Technology Testing Time base cellular engineering concept day design genome wide association study improved novel prototype radius bone structure research study response restriction enzyme self assembly simulation single molecule

项目摘要

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.

描述（由申请人提供）：当前确定染色体DNA上单核苷酸多态性（SNP）的方法需要微阵列（Affymetrix，Inc. -http：//www.affymetrix.com/index.com/index.affx; perlegen; perlegen科学 - http：/ （Opgen，Inc .- http：//www.opgen.com）。通常，这些方法在给定的测试中可以区分单个染色体上的几百个SNP，并且不会在超过1KBP上产生真正的单倍型信息。此外，DNA测序和微阵列技术受DNA杂交过程缓慢的限制。我们建议在流动设备（尤其是停滞点流）中的单个大型DNA分子的分离和拉伸方面使用最新进展，以基于序列 - 特异性杂交与荧光束结合的探针的快速，基因组测序（SNP和TAG SNP）技术开发快速的单分子，基因组测序（SNP和TAG 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并将处理时间减少到几个小时（每千SNP），从而彻底改变了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300K的标签SNP。因此，我们提出的主要问题是：是否可以通过利用DNA的微流体操纵来开发单个分子方法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与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“ dna，confemers？contemers”的这些方法来证明实现这一目标的技术？ - 噬菌体DNA，T4和大肠杆菌DNA，后者包括超过1 Mbp的基因组长度。我们建议使用微流体中的单分子技术来开发有机体基因组（包括人基因组）的完整TAG SNP扫描的技术。人类基因组的完整标签SNP扫描，在几天之内廉价且几天后完成，为个性化医学时代奠定了基础。基因组广泛的医学研究研究，包括早期预测疾病的遗传趋势以及对药物反应的基因组倾向，可以广泛使用，从而对药物基因组学领域产生巨大影响。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Fluorescent marker for direct detection of specific dsDNA sequences.

DOI：
10.1021/ac9019895
发表时间：
2009-12-15
期刊：
ANALYTICAL CHEMISTRY
影响因子：
7.4
作者：
Dylla-Spears, Rebecca;Townsend, Jacqueline E.;Sohn, Lydia L.;Jen-Jacobson, Linda;Muller, Susan J.
通讯作者：
Muller, Susan J.

Labeling DNA for single-molecule experiments: methods of labeling internal specific sequences on double-stranded DNA.