18] Analyzing fidelity of DNA polymerases

Abstract

Publisher Summary The first fidelity assays used synthetic templates composed of only one or two nucleotides, z Incorporation of radioactively labeled complementary and noncomplementary nucleotides was monitored and the error rate defined as the ratio of noncomplementary to total nucleotides was incorporated. The first assay that used a natural DNA template for fidelity measurements was the ΦX reversion assay. Assays are used to describe the base substitution fidelity of DNA polymerases, usually at a small number of template nucleotides. Mutations comprise 12 different substitution errors and several other types of mistakes occurring at many different sites within genes. To detect these, an assay is described here that monitors polymerase errors that inactivate the nonessential α-complementation activity of the lacZ gene in bacteriophage M13mp2. The method used to obtain a broad view of DNA polymerase fidelity for hundreds of different errors and the target can be manipulated to provide new sequences for examining specific hypotheses. The chapter discusses the experimental approach used to measure the fidelity of DNA synthesis by purified polymerases in vitro. A gapped M13mp2 substrate is constructed in which the single-strand gap contains the lacZ a-complementation target sequence, The DNA polymerase of choice is used for gap-filling synthesis. A portion of the reaction products is then analyzed by agarose gel electrophoresis to assure complete synthesis. Another aliquot of the reaction is introduced into competent Escherichia coli ceils and these are plated onto petri dishes containing the chromogenic indicator X-Gal and a lawn of E. coli α-complementation host ceils (CSH50).