[19] Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies

The fidelity of DNA synthesis depends on polymerase-primer template- nucleotide interactions that can alter fidelity at different locations along a primer-template molecule. An important biological consequence is the appearance of mutational hot and cold spots. Factors known to influence fidelity include base stacking interactions, base context effects including primer-template slippage, polymerase active site constraints, protonated or ionized nucleotide substrates, polymerase accessory factors, and mutagenic metals and carcinogens. The chapter describes a simple general assay to measure the fidelity of polymerase reactions at arbitrary positions on DNA or RNA template strands. Fidelity measurements are carried out by resolving single-stranded DNA differing in lengths by single nucleotides resulting from elongation of a 5-32P-labeled primer by polymerase using polyacrylamide gel electrophoresis (PAGE). The only requirement is that integrated gel band intensities, corresponding to primer elongation at a template target site and at adjacent template sites, can be quantified. Detailed protocols are provided for assaying fidelity at specific, arbitrarily chosen template sites. Assays are described for polymerases that possess Y-exonuclease activity and for those that do not. The chapter describes a special application of the gel assay for measuring nucleotide incorporation and bypass at abasic template lesions.