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（由申请人提供）：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。单元型或多个单核苷酸多态性 (SNP) 的阶段确定组被提议将遗传变异与特定疾病的风险联系起来。这项研究的长期目标是使用源自原子力显微镜 (AFM) 技术的探针，以低成本和高精度展示高通量 DNA 大小测定和单倍型分析。我们建议将微米级的快速探针电泳技术与新型纳米荧光检测方案相结合。我们最近演示了 AFM 探针表面的 DNA 电泳。在这个提案中，我们利用高电泳场来拉伸长 DNA 片段，因为它们沿着 AFM 探针的表面移动。 DNA 片段长度或与 DNA 链杂交的遗传信息是通过检测单个生物分子在 AFM 尖端窄端限定的有限体积中离散通过期间的荧光来获得的。 DNA 链两端均带有荧光标记，用于 DNA 大小测定，并与不同 SNP 位点的荧光标记杂交，用于 DNA 单倍型分析。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通过探针尖端末端的荧光分子。在 ANSOM 中，探针尖端的尖端产生纳米级的强激发光源。无需昂贵的 PCR 扩增即可探测通过标准方法分离人类基因组 DNA 获得的单分子。电泳过程的小规模可实现高分析通量。最后，在同一条长 DNA 链上探测多个 SNP 的能力应该会提高单倍型分析的准确性。该提案的具体目标是 i) 演示沿 AFM 探针表面电泳期间位于 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 ) 使用探针电泳和荧光末端标签来演示 DNA 大小测定，并研究可明确测量的 DNA 长度范围，最后，iv) 通过检测多个单倍体来演示基因分型和单倍型分析使用长 DNA 链上的杂交荧光标记进行核苷酸多态性 (SNP)。该项目提出了一种低成本、高通量的方法，将单倍型分析应用于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。