16] Use of M13 ping-pong vectors and T4 DNA polymerase in oligodeoxynucleotide-directed mutagenesis

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This chapter describes a new vector—M13B119—and an improved strategy for cyclic selection that is especially useful for creating multiple mutations with large deletions. Another new vector—M13K119W—is also used to demonstrate how the conditions for oligodeoxynucleotide-directed mutagenesis can be optimized. However, the use of different polymerases—including T4 polymerase, Sequenase and polIk—gives similar ratios of true mutants to spurious deletional mutants. The use of higher temperature helps to reduce the percentage of spurious deletional mutants by approximately 10-20%. Novel M13 cloning vectors with four overlapping copies of EcoK (M 13K 119W) and EcoB (M 13B 119) are designed for improving the efficiency of mutagenesis using oligodeoxynucleotides. These vectors complement the vector series with four tandem copies of EcoK, M13KllRX, which has been shown to be useful in the generation of a series of unidirectional deletional mutants by a mixture of oligodeoxyribonucleotides. With the combined use of T4 DNA polymerase and Eco K selection, a high efficiency of mutagenesis of up to 75% can be obtained for large deletions.