Ontology type: schema:ScholarlyArticle
2004-05-23
AUTHORSFarid J Ghadessy, Nicola Ramsay, François Boudsocq, David Loakes, Anthony Brown, Shigenori Iwai, Alexandra Vaisman, Roger Woodgate, Philipp Holliger
ABSTRACTDNA 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... »
PAGES755-759
http://scigraph.springernature.com/pub.10.1038/nbt974
DOIhttp://dx.doi.org/10.1038/nbt974
DIMENSIONShttps://app.dimensions.ai/details/publication/pub.1000250277
PUBMEDhttps://www.ncbi.nlm.nih.gov/pubmed/15156154
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