DNA size separation using an Atomic Force Microscope

描述(申请人提供)：最近完成的人类基因组测序产生了对高通量DNA测序方法的大量需求，这些方法在比较遗传学、遗传诊断和单核苷酸多态(SNP)检测中的应用。目前的毛细管电泳法的性能正在达到极限，需要创新的方案来显着增加DNA测序的吞吐量。这项研究的目的是利用原子力显微镜(AFM)探针在高速和低浓度下演示基于DNA大小的分离。假设是如果我们能将分离环境缩小到原子力显微镜探头(10微米长)的规模。我们将这一假设建立在1)AFM针尖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针尖驱动分子的方式，2)通过具有尺寸限制产生的新特性的特定定制介质来分离DNA片段，以及3)通过AFM检测沉积在衬底上的DNA片段。其具体目标是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DNA片段长度的范围来展示该方法在量化DNA片段长度方面的分辨率限制，3)使用光学荧光检测来确定分析物的最小浓度，以及4)不同长度的特定DNA片段组的大小分离；第一步是纳米级DNA大小分离。我们预计，基于AFM的电泳粒度分离将比毛细管电泳法快三到四个数量级的吞吐量。此外，AFM操作的固有样品大小允许在意外的样品浓度接近单分子规模的情况下进行测量。

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.