Introducing Predetermined Mutations Throughout a Target Gene Using TDEM (Transposon-Directed Base-Exchange Mutagenesis)

Abstract

Transposon-directed base-exchange mutagenesis (TDEM) is an efficient and controllable method for introducing a mutation(s) into a gene. Each round of TDEM removes a predetermined number of bases (up to 11 base pairs) from a randomly selected site within the target gene and replaces them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be precisely regulated. Because each round of TDEM generates mutation(s) at a single site, the number of mutations introduced can be determined by the number of cycles of TDEM. Furthermore, using a novel frame-checking procedure, non-functional mutants containing a frameshift or stop codon can be minimized. Thus, TDEM can be used to introduce a limited and predetermined change at each round of mutagenesis, thereby providing a useful tool for studying protein structure and function.