DNA size separation using an Atomic Force Microscope

使用原子力显微镜分离 DNA 大小

• 批准号: 7114737
• 负责人: Hemantha Kumar Wickramasinghe
• 金额: $ 15.25万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7114737
• 关键词: DNA; genetics; motivation

DESCRIPTION (provided by applicant): The recent completion of the human genome sequencing has generated a large demand for high throughput DNA sequencing methods with applications in comparative genetics, genetic diagnosis and Single Nucleotide Polymorphisms (SNP) detection. Current capillary electrophoretic methods are reaching a limit in their performance and innovative schemes are required for a significant increase in the DNA sequencing throughput. The goal of this research is to demonstrate DNA size-based separation at high speed and low concentration using Atomic Force Microscopy (AFM) probes. The hypothesis is if we can scale the separation environment down to the scale of an AFM probe (10 micrometer long). We base this hypothesis on 1) AFM tips as a medium for electrophoretic size separation: an electric field is applied in a manner almost parallel to the surface of the probe to drive the molecules along the tip, 2) size separation of DNA fragments by specifically tailored medium with new properties arising from size confinement and 3) detection of DNA fragments deposited on a substrate by AFM. The specific aims are to 1) determine the electrophoretic mobility of DNA fragments in the medium of the surface of an AFM probe, 2) to demonstrate the limits in the resolution of this method to quantify DNA fragment lengths by investigating a range of DNA fragment lengths, 3) determine the minimal concentration of analytes using optical fluorescence detection and 4) size separation of specific DNA fragment groups with different lengths; in a first step to nanometer scale DNA size separation. We anticipate that AFM based electrophoretic size separation will reach a throughput of three to four order of magnitudes faster than capillary electrophoresis methods. Furthermore, the inherent sample size on which the AFM operates allows measurements to be performed with an unforeseen specimen concentration approaching the single molecule scale.

描述（由申请人提供）：人类基因组测序的最新完成产生了对高通量DNA测序方法的巨大需求，其应用于比较遗传学、基因诊断和单核苷酸多态性（SNP）检测。当前的毛细管电泳方法的性能已达到极限，需要创新方案来显着提高 DNA 测序通量。本研究的目标是展示使用原子力显微镜 (AFM) 探针在高速和低浓度下进行基于 DNA 大小的分离。假设我们是否可以将分离环境缩小到 AFM 探针的尺寸（10 微米长）。我们的这一假设基于 1) AFM 尖端作为电泳尺寸分离的介质：以几乎平行于探针表面的方式施加电场，以沿着尖端驱动分子，2) 通过特定的方法对 DNA 片段进行尺寸分离定制的介质具有由于尺寸限制而产生的新特性，以及 3) 通过 AFM 检测沉积在基底上的 DNA 片段。具体目标是 1) 确定 DNA 片段在 AFM 探针表面介质中的电泳迁移率，2) 通过研究一系列 DNA 片段长度来证明这种定量 DNA 片段长度方法的分辨率限制, 3) 使用光学荧光检测确定分析物的最低浓度，4) 不同长度的特定 DNA 片段组的大小分离；纳米级 DNA 尺寸分离的第一步。我们预计基于 AFM 的电泳尺寸分离的通量将比毛细管电泳方法快三到四个数量级。此外，AFM 操作的固有样本大小允许以接近单分子规模的不可预见的样本浓度进行测量。