Molecular breeding of polymerases for resistance to environmental inhibitors

Claudia Baar; Marc d'Abbadie; Alexandra Vaisman; Mercedes E. Arana; Michael Hofreiter; Roger Woodgate; Thomas A. Kunkel; Philipp Holliger

doi:10.1093/nar/gkq1360

Molecular breeding of polymerases for resistance to environmental inhibitors

Claudia Baar, Marc d'Abbadie, Alexandra Vaisman, Mercedes E. Arana, Michael Hofreiter, Roger Woodgate, Thomas A. Kunkel, Philipp Holliger

Biology

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Article number	e51
Pages (from-to)	-
Number of pages	12
Journal	Nucleic Acids Research
Volume	39
Issue number	8
DOIs	https://doi.org/10.1093/nar/gkq1360
Publication status	Published - Apr 2011

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AU - d'Abbadie, Marc

AU - Vaisman, Alexandra

AU - Arana, Mercedes E.

AU - Hofreiter, Michael

AU - Woodgate, Roger

AU - Kunkel, Thomas A.

AU - Holliger, Philipp

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Molecular breeding of polymerases for resistance to environmental inhibitors

Abstract

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