DNA size separation using an Atomic Force Microscope

描述（由申请人提供）：最近人类基因组测序的完成对高通量DNA测序方法产生了大量需求，应用于比较遗传学，遗传诊断和单核苷酸多态性（SNP）检测。目前的毛细管电泳方法在性能上已经达到了极限，需要创新的方案来显著提高DNA测序的通量。本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。假设我们可以将分离环境缩小到AFM探针的规模（10微米长）。我们基于以下假设：1)AFM尖端作为电泳尺寸分离的介质：电场以几乎平行于探针表面的方式施加以驱动分子沿着尖端；2)通过具有尺寸限制产生的新特性的专门定制的介质分离DNA片段；3)通过AFM检测沉积在底物上的DNA片段。具体目的是1)确定AFM探针表面介质中DNA片段的电泳迁移率，2)通过研究DNA片段长度范围来证明该方法定量DNA片段长度的分辨率限制，3)使用光学荧光检测确定分析物的最小浓度，4)不同长度的特定DNA片段群的大小分离；这是迈向纳米级DNA分离的第一步。我们预计，基于AFM的电泳尺寸分离将达到比毛细管电泳方法快三到四个数量级的吞吐量。此外，AFM操作的固有样本量允许在接近单分子尺度的不可预见的样品浓度下进行测量。

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.