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）：最近的人类基因组测序的最近完成对高吞吐量DNA测序方法的需求很大，并在比较遗传学，遗传诊断和单核苷酸多态性（SNP）检测中施用。当前的毛细管电泳方法达到了其性能的极限，并且需要创新的方案才能显着增加DNA测序吞吐量。这项研究的目的是使用原子力显微镜（AFM）探针在高速和低浓度下基于DNA大小的分离。假设是我们是否可以将分离环境扩展到AFM探针的尺度（长10千分尺）。我们以1）为基于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平行地应用电场，以沿尖端驱动分子，2）通过特异性地定制了DNA片段的尺寸分离，该尺寸量身定制，该介质具有由尺寸约束而产生的新特性和3）在dna片段上的新特性，该介质在dna片段上沉积了dna碎片。具体目的是1）确定DNA片段在AFM探针表面介质中的电泳迁移率，2），以证明该方法解决方案中的限制，以通过研究分析的最小荧光尺寸分离的分析范围来量化DNA片段长度，以研究DNA片段长度，3）。在纳米尺度的第一步中，DNA尺寸分离。我们预计，基于AFM的电泳尺寸分离将比毛细管电泳方法快三到四个大小的吞吐量。此外，AFM操作的固有样本量允许以不可预见的标本浓度接近单分子尺度进行测量。