DNA Repair Proficient PCR

Award Information
Agency:
Department of Health and Human Services
Branch
n/a
Amount:
$99,050.00
Award Year:
2009
Program:
SBIR
Phase:
Phase I
Contract:
1R43GM088954-01
Agency Tracking Number:
GM088954
Solicitation Year:
n/a
Solicitation Topic Code:
n/a
Solicitation Number:
n/a
Small Business Information
LUCIGEN CORPORATION
2120 W. GREENVIEW DR., SUITE 9, MIDDLETON, WI, 53562
Hubzone Owned:
N
Socially and Economically Disadvantaged:
N
Woman Owned:
N
Duns:
019710669
Principal Investigator:
JEFFREY WILLIAMS
() -
JWILLIAMS@LUCIGEN.COM
Business Contact:
DAVID MEAD
() -
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 particu lar, 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 terminat es. 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 pyro phosphatase. 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 medici ne 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 yie ld, sensitivity, and throughput of DNA-based diagnostics. PDF Creator - PDF4Free v2.0 http://www.pdf4free.com

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