Molecular breeding of polymerases for resistance to environmental inhibitors

Potent inhibitors limit the use of PCR assays in a wide spectrum of specimens. Here, we describe the engineering of polymerases with a broad resistance to complex environmental inhibitors using molecular breeding of eight different polymerase orthologues from the genus Thermus and directed evolution...

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Bibliographic Details
Published in:Nucleic acids research 2011-04, Vol.39 (8), p.e51-e51
Main Authors: Baar, Claudia, d'Abbadie, Marc, Vaisman, Alexandra, Arana, Mercedes E, Hofreiter, Michael, Woodgate, Roger, Kunkel, Thomas A, Holliger, Philipp
Format: Article
Language:English
Subjects:
Bone and Bones
Directed Molecular Evolution - methods
DNA-Directed DNA Polymerase - chemistry
DNA-Directed DNA Polymerase - genetics
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - pharmacology
Gene Library
Humic Substances
Methods Online
Polymerase Chain Reaction
Soil
Taq Polymerase
Thermus
Thermus - enzymology
Thermus aquaticus
Thermus brockianus
Thermus thermophilus
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Summary:Potent inhibitors limit the use of PCR assays in a wide spectrum of specimens. Here, we describe the engineering of polymerases with a broad resistance to complex environmental inhibitors using molecular breeding of eight different polymerase orthologues from the genus Thermus and directed evolution by CSR in the presence of inhibitors. Selecting for resistance to the inhibitory effects of Neomylodon bone powder, we isolated 2D9, a chimeric polymerase comprising sequence elements derived from DNA polymerases from Thermus aquaticus, Thermus oshimai, Thermus thermophilus and Thermus brockianus. 2D9 displayed a striking resistance to a broad spectrum of complex inhibitors of highly divergent composition including humic acid, bone dust, coprolite, peat extract, clay-rich soil, cave sediment and tar. The selected polymerase promises to have utility in PCR-based applications in a wide range of fields including palaeobiology, archaeology, conservation biology, forensic and historic medicine.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkq1360