期刊
NUCLEIC ACIDS RESEARCH
卷 39, 期 8, 页码 -出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkq1360
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资金
- MRC (Medical Research Council) [MC_US_A024_0014]
- DFG (Deutsche Forschungsgemeinschaft) [BA 3572/1-1]
- MRC
- Trinity College, Cambridge
- NICHD (National Institute of Child Health and Human Development)/NIH
- Division of Intramural Research of the NIH, NIEHS (National Institute of Environmental Health Sciences) [Z01 ES065070]
- Medical Research Council [MC_U105178804] Funding Source: researchfish
- MRC [MC_U105178804] Funding Source: UKRI
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.
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