REPAIR-RESISTANT NUCLEOSIDE ANALOGUES IN OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS

Abstract

2'-Deoxytubercidin (a) and 2'-deoxy-7-deazainosine (b) were used as analogues of deoxyadenosine and deoxyinosine respectively for the introduction of transition mutations into the a-peptide fragment of the lacZ gene encoded in the filamentous bacteriophage Ml13mp9. Using conventional methodology of oligonucleotide-directed mutagenesis, the number of desired point mutants was very low (2-6%) for both T+C and C+T transitions, due to the efficient removal of purine/pyrimidine mismatches by the repair machinery of E. coli. Following the incorporation (via synthetic oligodeoxyribonucleotides) of the purine nucleoside analogues a and b into the mismatch sites, the yield of desired mutants was increased approximately tenfold.