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doi: 10.1038/nbt974. Epub 2004 May 23. Affiliations
Generic expansion of the substrate spectrum of a DNA polymerase by directed evolutionFarid J Ghadessy et al. Nat Biotechnol. 2004 Jun. AbstractDNA 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. Similar articles
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For which enzyme are nucleotides the substrate?Antibody–nucleotide conjugate as a substrate for DNA polymerases.
What does DNA polymerase do Labster?DNA polymerase is the enzyme that is responsible for the formation of a DNA strand from a template DNA strand. For the DNA polymerase to initiate the formation of a new DNA strand, it needs a short "starter" strand, called a Primer. The primer must be bound to an area of the template DNA to which it is complementary.
What type of nucleotides are used in PCR?Deoxynucleoside triphosphates (dNTPs) dNTPs consist of four basic nucleotides—dATP, dCTP, dGTP, and dTTP—as building blocks of new DNA strands. These four nucleotides are typically added to the PCR reaction in equimolar amounts for optimal base incorporation.
Which type of enzyme is used in PCR technology?Taq polymerase
Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus).
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For which enzyme are nucleotides the substrate labster
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