DNA Repair Proficient PCR

Award Information
Agency: Department of Health and Human Services
Branch: N/A
Contract: 1R43GM088954-01
Agency Tracking Number: GM088954
Amount: $99,050.00
Phase: Phase I
Program: SBIR
Awards Year: 2009
Solicitation Year: 2009
Solicitation Topic Code: N/A
Solicitation Number: PHS2009-2
Small Business Information
2120 W. GREENVIEW DR., SUITE 9, MIDDLETON, WI, 53562
DUNS: 019710669
HUBZone Owned: Y
Woman Owned: N
Socially and Economically Disadvantaged: N
Principal Investigator
 JEFFREY WILLIAMS
 () -
 JWILLIAMS@LUCIGEN.COM
Business Contact
 DAVID MEAD
Phone: (608) 831-9011
Email: dmead@lucigen.com
Research Institution
N/A
Abstract
DESCRIPTION (provided by applicant): The Polymerase Chain Reaction (PCR) is one of the most widely used techniques in molecular biology. Unfortunately, thermocycling at elevated temperatures (60o to 95oC) causes extensive thermal damage to DNA; in particular, A+G depurination. In vivo, apurinic sites are repaired by AP lyase enzymes, which cleave DNA to form elongatable 3'OH ends. However, in vitro PCR reactions generally lack DNA repair enzymes. Since apurinic sites are not removed, DNA synthesis terminates. In this Phase I Research grant application, enzymology is described for DNA repair- proficient PCR. A novel thermostable enzyme mixture will be developed, using recombinant Thermotoga maritima AP lyase, PyroPhage 3173 polymerase, and an engineered pyrophosphatase. In DNA repair-proficient PCR, sites of A+G depurination are enzymatically hydrolysed to 3'OH ends during thermocycling, so that thermally damaged DNA strands are continually converted into elongatable molecules. As a result, it is hypothesized that the yield, sensitivity, and amplicon length of PCR can be increased. PDF Creator - PDF4Free v2.0 http://www.pdf4free.com PUBLIC HEALTH RELEVANCE: The Polymerase Chain Reaction (PCR) is one of the most widely used techniques in molecular medicine for the diagnosis of heritable and infectious diseases. However, the high-temperature reaction conditions employed during PCR result in thermal damage to DNA. The proposed research describes a repair-proficient enzyme mixture to improve the reaction yield, sensitivity, and throughput of DNA-based diagnostics. PDF Creator - PDF4Free v2.0 http://www.pdf4free.com

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