1 R21 R21HG04549
Rapid DNA Sizing and Haplotyping Using Probe Electophore
| Principal Investigator: |
Hemantha Wickramasinghe
University of California, Irvine
The Henry Samueli School of Engi
University of California, Irvine
|
Project Period: 06/01/2008 - 05/31/2010
Abstract (from grant application):
DESCRIPTION (provided by applicant): The completion of the human genome project provided a complete template for the systematic study of the human genetic variants. Haplotypes or the phase-determined sets of multiple Single Nucleotide Polymorphisms (SNPs) are proposed to link genetic variants to the risk for specific illnesses. The long-term goal of this research is to demonstrate high throughput DNA sizing and haplotyping at low cost and high accuracy using probes derived from the Atomic Force Microscopy (AFM) techniques. We propose to combine a rapid probe electrophoresis technique at the micrometer scale with a novel nanometer fluorescence detection scheme. We recently demonstrated DNA electrophoresis at the surface of an AFM probe. In this proposal, we utilize the high electrophoretic field to stretch long DNA fragments, as they move along the surface of the AFM probe. The DNA fragment length or the genetic information hybridized to DNA strands are obtained by detecting the fluorescence during the discrete passage of single bio-molecules in the confined volume defined by the narrow end of an AFM tip. DNA strands are fluorescently tagged at both ends for DNA sizing and are hybridized with fluorescent markers at different SNP sites for DNA haplotyping. Fluorescent molecules passing the end of the probe tip are detected by adapting the apertureless scanning near field optical microscopy (ANSOM) scheme into a non- scanning configuration. In ANSOM, the sharp end of a probe tip creates a nanometer scale strong source of excitation light. Single molecules obtained from standard methods to isolate human genomic DNA are probed without costly PCR amplification. The small scale of the electrophoresis process allows for high analysis throughputs. Finally, the ability to probe multiple SNPs on the same long DNA strand should result in a higher accuracy for haplotyping. The specific aims of this proposal are i) to demonstrate the excitation and detection of fluorophores located at different positions on a DNA fragment during electrophoresis along the surface of an AFM probe, ii) to calibrate and determine the resolution of the proposed detection method, iii) to demonstrate DNA sizing using probe electrophoresis and fluorescent end tags and investigate the range of unambiguously measurable lengths of DNA and finally, iv) to demonstrate genotyping and haplotyping by detecting multiple single nucleotide polymorphisms (SNP) using hybridized fluorescent marker labels on long DNA strands. This project proposes a low cost and high throughput method bringing haplotyping into practical use in the field of personal medicine. Further applications will have a key impact in genetics, drug design, epidemiology, and in evolution studies.
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