title: Molecular breeding of polymerases for resistance to environmental inhibitors.
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.
authors.affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
authors.forename: Claudia
authors.initials: C
authors.lastname: Baar
authors.forename: Marc
authors.initials: M
authors.lastname: d'Abbadie
authors.forename: Alexandra
authors.initials: A
authors.lastname: Vaisman
authors.forename: Mercedes E
authors.initials: ME
authors.lastname: Arana
authors.forename: Michael
authors.initials: M
authors.lastname: Hofreiter
authors.forename: Roger
authors.initials: R
authors.lastname: Woodgate
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kunkel
authors.forename: Philipp
authors.initials: P
authors.lastname: Holliger
mesh_terms: D001842:Bone and Bones; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D015723:Gene Library; D006812:Humic Substances; D016133:Polymerase Chain Reaction; D012987:Soil; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D006812:Humic Substances; D012987:Soil; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase