Misincorporation by AMV reverse transcriptase and its potential for mutagenesis.
thesisposted on 19.11.2015, 09:07 by Judith Ann. Skinner
This thesis describes a systematic investigation of the efficiency of misincorporation by Avian Myeloblastosis Virus reverse transcriptase with all possible combinations of dNTP substrate, template nucleotide, and the nucleotide at the 3' terminus of the primer. Each of a series of 16 synthetic oligonucleotide primers was annealed to single-stranded Ml3 DNA templates, and a single dNTP was misincorporated at the primer 3' end using AMV reverse transcriptase. The proportion and pattern of misincorporation and incorporation in all 64 situations were assayed using 5'-labelled primers, and the products were separated on denaturing polyacrylamide gels. Correct incorporations occurred more readily than misincorporations. The efficiency of misincorporation depended on the individual primer but, comparing primers, a clear dependence on the template nucleotide was observed for the preferential misincorporation of different dNTPs. The exact combination of template nucleotide and dNTP was important; although purine:pyrimidine (dNTP substrate: template nucleotide) and pyrimidine:purine misincorporations occurred comparatively readily, some pyrimidine:pyrimidine and purine:purine reactions were equally efficient and yet others were never seen to occur. Some misincorporations were facilitated by subsequent correct incorporations, but despite this the results suggest that the level of misincorporation is limited by the rate of reaction and enzyme inactivation rather than by exonuclease activity. The recovery of point mutants arising from reverse transcriptase-directed misincorporation of single dNTPs onto single oligonucleotide primers is described and discussed. Misincorporation of dNTPs is a form of in vitro mutagenesis which facilitates the generation of a library of point mutations spread throughout a gene. Conditions have been established in this study for the production of a bank of primers with 3' termini distributed over a region of a gene to be mutated. The misincorporation of single dNTPs onto the termini of such a bank of primers should allow the generation of a library of point mutants.