Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2004-05-23

AUTHORS

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

ABSTRACT

DNA polymerases recognize their substrates with exceptionally high specificity1,2, 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-nitroindol3 and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates4 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. More... »

PAGES

755-759

Journal

TITLE

Nature Biotechnology

ISSUE

6

VOLUME

22

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nbt974

DOI

http://dx.doi.org/10.1038/nbt974

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1000250277

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/15156154


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