Research Project
7287868
DESCRIPTION (provided by applicant): Whole genome amplification by the multiple displacement amplification (MDA) reaction has the potential to transform the way that DNA is prepared for genotyping assays. The long-term goal is to develop a low cost and rapid process for amplifying genomic DNA directly from minute clinical specimens or stored DNA samples. DNA amplified from blood and buccal swab samples will be ready for use in SNP assays, bypassing conventional bead or cartrige based DNA purification steps. New research applications of MDA will be investigated based on its ability to amplify DNA from single cells. The specific aims are to: 1. Optimize the MDA reaction enzymology. Molecular mechanisms will be investigated for the "hyperbranching" process that underlies exponential amplification. Reaction intermediates will be evaluated for the steps of template denaturation, random priming, and strand displacement DNA synthesis. Parameters will be defined and measures that affect reaction rate, DNA yield, amplification bias, and fidelity of sequence replication. 2. Develop a low cost, high throughput DNA sample preparation process for use in SNP genotyping. An integrated process will deliver DNA from blood and buccal swab samples directly into genotyping assays. Miniaturization and microfluidic methods will add to throughput and cost reduction. Processes will be validated for DNA preparation from archives of biological samples and rare DNA collections to increase access to a wider range of researchers. 3. Demonstrate methods for whole genome amplification from single cells. A) Single sperm will be used as a means to obtain haplotypes. B) Cells isolated from human tumors will be assessed for genetic heterogeneity in the transformation process. C) Individual lymphocytes will be used as a model system to prove feasibility of doing preimplantation genetic diagnosis from single blastomeres.
