Rapid DNA Sizing and Haplotyping Using Probe Electophoresis

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。单倍型或阶段决定的多单核苷酸多态性（snp）被提出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，以低成本和高精度证明高通量DNA大小和单倍型。我们提出将一种微米尺度的快速探针电泳技术与一种新的纳米荧光检测方案相结合。我们最近展示了AFM探针表面的DNA电泳。在这个建议中，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由AFM尖端窄端定义的受限体积内离散通过时的荧光来获得的。DNA链在两端进行荧光标记以确定DNA大小，并与不同SNP位点的荧光标记进行杂交以确定DNA单倍型。通过探针末端的荧光分子被检测通过无孔径扫描近场光学显微镜（ANSOM）方案，以非扫描配置。在ANSOM中，探针尖端的尖端产生纳米级的强激发光源。从标准方法分离人类基因组DNA获得的单分子不需要昂贵的PCR扩增。小规模的电泳过程允许高分析吞吐量。最后，在同一长DNA链上探测多个snp的能力应该会导致单倍型的更高准确性。本提案的具体目的是i)演示沿AFM探针表面电泳过程中位于DNA片段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演示DNA大小，并研究DNA长度明确可测量的范围，最后，iv)利用杂交荧光标记在长DNA链上检测多个单核苷酸多态性（SNP），证明基因分型和单倍分型。本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。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。

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.