Hypermutagenic in vitro transcription employing biased NTP pools and manganese cations.
In vitro DNA-dependent RNA transcription using bacteriophage T3 RNA polymerase may be rendered hypermutagenic by employing biased nucleoside triphosphate (NTP) concentrations and manganese cations. Using the E. coli R67 plasmid-encoded dihydrofolate reductase (DHFR) gene as target substitution rates approaching 4 x 10(-2) per base per reaction could be achieved, on a par with hypermutagenic reverse transcription. In all cases the majority of substitutions was that expected from the NTP pool bias. The addition of manganese ions increased the frequency of mutations, particularly the proportion of transversions. Functional DHFR hypermutants with up to 8% amino acid substitutions were readily obtained from a single reaction which, given the unique mutation matrix allows exploration of sequence space complementary to that accessed by other hypermutagenic protocols.
Pezo
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Unité de Rétrovirologie Moléculaire, Institut Pasteur, Paris, France.
Wain-Hobson
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D000255:Adenosine Triphosphate / Q000235:genetics; D000595:Amino Acid Sequence; D000667:Ampicillin / Q000494:pharmacology; D002412:Cations / Q000737:chemistry; D003570:Cytidine Triphosphate / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000235:genetics; D008345:Manganese / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009705:Nucleosides / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014295:Trimethoprim / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000235:genetics
D002412:Cations; D009705:Nucleosides; D008345:Manganese; D003570:Cytidine Triphosphate; D000667:Ampicillin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; C081591:bacteriophage T3 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate