Rapid DNA Sizing and Haplotyping Using Probe Electophoresis

使用探针电泳进行快速 DNA 大小测定和单元分型

• 批准号: 7365300
• 负责人: Hemantha Kumar Wickramasinghe
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7365300
• 关键词: Atomic Force Microscopy; Chemistry; Classification; Coupling; DNA; DNA Probes; Detection; Drug Design; Electrophoresis; Epidemiology; Evolution; Fluorescence; Genetic; Genomics; Genotype; Goals; Haplotypes; Human; Human Genome Project; Label; Length; Light; Link; Location; Measurable; Measures; Medicine; Methods; Microscope; Microscopy; Optics; Phase; Polymerase Chain Reaction; Positioning Attribute; Principal Investigator; Process; Range; Reading; Research; Resolution; Risk; Scanning; Scheme; Single Nucleotide Polymorphism; Site; Source; Standards of Weights and Measures; Stretching; Surface; Techniques; Time; Work; base; concept; cost; falls; fluorophore; genetic variant; high throughput analysis; nanometer; nanoscale; novel; optical imaging; research study; single molecule; size

DESCRIPTION (provided by applicant): The completion of the human genome project provided a complete template for the systematic study of the human genetic variants. Haplotypes or the phase-determined sets of multiple Single Nucleotide Polymorphisms (SNPs) are proposed to link genetic variants to the risk for specific illnesses. The long-term goal of this research is to demonstrate high throughput DNA sizing and haplotyping at low cost and high accuracy using probes derived from the Atomic Force Microscopy (AFM) techniques. We propose to combine a rapid probe electrophoresis technique at the micrometer scale with a novel nanometer fluorescence detection scheme. We recently demonstrated DNA electrophoresis at the surface of an AFM probe. In this proposal, we utilize the high electrophoretic field to stretch long DNA fragments, as they move along the surface of the AFM probe. The DNA fragment length or the genetic information hybridized to DNA strands are obtained by detecting the fluorescence during the discrete passage of single bio-molecules in the confined volume defined by the narrow end of an AFM tip. DNA strands are fluorescently tagged at both ends for DNA sizing and are hybridized with fluorescent markers at different SNP sites for DNA haplotyping. Fluorescent molecules passing the end of the probe tip are detected by adapting the apertureless scanning near field optical microscopy (ANSOM) scheme into a non- scanning configuration. In ANSOM, the sharp end of a probe tip creates a nanometer scale strong source of excitation light. Single molecules obtained from standard methods to isolate human genomic DNA are probed without costly PCR amplification. The small scale of the electrophoresis process allows for high analysis throughputs. Finally, the ability to probe multiple SNPs on the same long DNA strand should result in a higher accuracy for haplotyping. The specific aims of this proposal are i) to demonstrate the excitation and detection of fluorophores located at different positions on a DNA fragment during electrophoresis along the surface of an AFM probe, ii) to calibrate and determine the resolution of the proposed detection method, iii) to demonstrate DNA sizing using probe electrophoresis and fluorescent end tags and investigate the range of unambiguously measurable lengths of DNA and finally, iv) to demonstrate genotyping and haplotyping by detecting multiple single nucleotide polymorphisms (SNP) using hybridized fluorescent marker labels on long DNA strands. This project proposes a low cost and high throughput method bringing haplotyping into practical use in the field of personal medicine. Further applications will have a key impact in genetics, drug design, epidemiology, and in evolution studies.

描述（申请人提供）：人类基因组计划的完成为人类遗传变异的系统研究提供了完整的模板。单倍型或多个单核苷酸多态性（SNP）的阶段确定集被提议将遗传变异与特定疾病的风险联系起来。本研究的长期目标是使用原子力显微镜（AFM）技术衍生的探针以低成本和高准确度展示高通量DNA大小测定和单体型分析。我们提出将微米级的快速探针电泳技术与新型的纳米荧光检测方案联合收割机相结合。我们最近展示了DNA电泳在AFM探针的表面。在这个提议中，我们利用高电泳场拉伸长DNA片段，因为它们沿着AFM探针的表面移动。通过检测单个生物分子在由AFM针尖的窄端限定的受限体积中离散通过期间的荧光来获得与DNA链杂交的DNA片段长度或遗传信息。DNA链在两端被荧光标记用于DNA大小测定，并在不同的SNP位点与荧光标记杂交用于DNA单倍型分析。通过探针末端的荧光分子通过将无孔扫描近场光学显微镜（ANSOM）方案调整为非扫描配置来检测。在ANSOM中，探针尖端的尖端产生纳米级的强激发光源。从分离人类基因组DNA的标准方法获得的单分子被探测，而不需要昂贵的PCR扩增。电泳过程的小规模允许高分析通量。最后，在同一长DNA链上探测多个SNP的能力应该会导致更高的单体型分析准确性。该提议的具体目的是i）证明在沿着AFM探针的表面电泳期间位于DNA片段上不同位置的荧光团的激发和检测，ii）校准和确定所提出的检测方法的分辨率，iii）证明使用探针电泳和荧光末端标签的DNA大小测定，并研究DNA的明确可测量长度的范围，以及最后，iv）通过使用长DNA链上的杂交荧光标记物标记检测多个单核苷酸多态性（SNP）来证明基因分型和单体型分型。该项目提出了一种低成本和高通量的方法，使单倍型在个人医学领域的实际应用。进一步的应用将对遗传学、药物设计、流行病学和进化研究产生关键影响。