HYPERMUTAGENIC IN-VITRO TRANSCRIPTION EMPLOYING BIASED NTP POOLS AND MANGANESE CATIONS

In vitro DNA-dependent RNA transcription using bacteriophage T3 RNA po lymerase 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 targe t substitution rates approaching 4 x 10(-2) per base per reaction coul d 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 DH FR hypermutants with up to 8% amino acid substitutions were readily ob tained from a single reaction which, given the unique mutation matrix allows exploration of sequence space complementary to that accessed by other hypermutagenic protocols.