Rapid DNA Sizing and Haplotyping Using Probe Electophoresis

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一个完整的模板。单倍型或多个单核苷酸多态(SNPs)的阶段决定集(SNPs)被提出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来自原子力显微镜(AFM)技术的探针以低成本和高精度展示高通量DNA大小和单倍型。我们建议将微米级的快速探针电泳技术与新的纳米荧光检测方案相结合。我们最近展示了原子力显微镜探针表面的DNA电泳法。在这个方案中，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针尖窄端限定的受限体积内离散通过时的荧光来获得的。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。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的荧光分子。在安索姆，探测器尖端的尖端产生了一个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。由于电泳法的规模较小，因此可以获得较高的分析产量。最后，在同一条长DNA链上探测多个SNP的能力应该会带来更高的单倍型准确性。这项建议的具体目标是：i)展示沿AFM探针表面的电泳过程中位于DNA片段上不同位置的荧光团的激发和检测；ii)校准和确定所提议的检测方法的分辨率；iii)利用探针电泳和荧光末端标签展示DNA的大小并调查明确可测量的DNA长度的范围；以及iv)通过使用长DNA链上的杂交荧光标记检测多个单核苷酸多态性(SNP)来展示基因分型和单倍化。本项目提出了一种低成本、高通量的方法，将单倍体分析技术应用于个人医学领域。进一步的应用将在遗传学、药物设计、流行病学和进化论研究中产生关键影响。

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.