DNA size separation using an Atomic Force Microscope

University Of California-Irvine, Irvine CA

Investigators

Abstract

[unreadable] 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.[unreadable] [unreadable] [unreadable]