Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution.

Farid J Ghadessy, Nicola Ramsay, François Boudsocq, David Loakes, Anthony Brown, Shigenori Iwai, Alexandra Vaisman, Roger Woodgate, Philipp Holliger

文献索引：Nat. Biotechnol. 22(6) , 755-9, (2004)

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摘要

DNA polymerases recognize their substrates with exceptionally high specificity, restricting the use of unnatural nucleotides and the applications they enable. We describe a strategy to expand the substrate range of polymerases. By selecting for the extension of distorting 3' mismatches, we obtained mutants of Taq DNA polymerase that not only promiscuously extended mismatches, but had acquired a generic ability to process a diverse range of noncanonical substrates while maintaining high catalytic turnover, processivity and fidelity. Unlike the wild-type enzyme, they bypassed blocking lesions such as an abasic site, a thymidine dimer or the base analog 5-nitroindol and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates or the substitution of one with the equivalent fluorescent dye-labeled nucleotide triphosphate. Such 'unfussy' polymerases have immediate utility, as we demonstrate by the generation of microarray probes with up to 20-fold brighter fluorescence.