Blood Inhibition Resistant Mutants of Taq DNA Polymerase

Award Information
Agency: Department of Health and Human Services
Branch: N/A
Contract: 2R44GM073401-02
Agency Tracking Number: GM073401
Amount: $678,280.00
Phase: Phase II
Program: SBIR
Awards Year: 2006
Solicitation Year: 2006
Solicitation Topic Code: N/A
Solicitation Number: PHS2006-2
Small Business Information
DNA POLYMERASE TECHNOLOGY, INC., 1508 S GRAND BLVD, ST. LOUIS, MO, 63104
DUNS: N/A
HUBZone Owned: N
Woman Owned: N
Socially and Economically Disadvantaged: N
Principal Investigator
 MILKO KERMEKCHIEV
 (314) 771-5566
 MILKO@KLENTAQ.COM
Business Contact
 WAYNE BARNES
Phone: (314) 771-5566
Email: WAYNE@KLENTAQ.COM
Research Institution
N/A
Abstract
DESCRIPTION (provided by applicant): The sensitivity of gene detection in blood specimens in important clinical and forensic PCR applications is limited due to inhibitory blood substances which reduce amplification efficiency and may lead to false negative results. Different pre-PCR treatments that are being used to overcome this inhibitory effect significantly increase the duration and cost of the tests. Our project started with the idea to use the in vitro evolution approach to develop KlenTaq polymerase mutants that are highly resistant to the blood inhibition, which will allow a faster and lower-cost direct PCR analysis of blood samples. We also proposed to select KlenTaq mutants with rapid DMA elongation, which will reduce the time of the amplification cycle. Both types of mutant enzymes were obtained and characterized in the Phase I of the project. KlenTaq mutants combining the two selected phenotypes were also obtained. In Phase II we propose to further evolve the obtained mutant enzymes by determining the best amino acid substitutions at the codons responsible for the above mutations. To this end, we will use saturating site- directed mutagenisis. Next, using the same technique, we plan to transfer these amino acid changes into the full-length Taq enzyme, thus expanding the scope of PCR applications. Finally we will try to combine the two novel qualities of Taq with the cold-sensitive mutants of the enzyme designed for highly specific hot-start PCR already achieved by our company. The resultant triple-quality enzymes will be included in PCR kits specialized for detection of clinically important targets including HIV and Hepatitis B. Until now, the diagnosis of infectious diseases and genetic disorders has required costly and time- consuming procedures. We believe our new products will provide improved accuracy, efficiency, and lower cost of these tests, which will benefit the public by making them more affordable in the U.S. and worldwide.

* Information listed above is at the time of submission. *

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