A WALK-THROUGH TECHNIQUE FOR IN VITRO RECOMBINATION OF POLYNUCLEOTIDE SEQUENCES

摘要

A simple and efficient method for random in vitro mutagenesis and recombination of polynucleotide sequences based on walk-through with chain- elongating molecules and chain terminating molecules e.g. dNTPs/ddNTPs followed by reassembly and amplification is described. The utility of this method for improving protein structure and/or function is demonstrated by creating novel sarcosine oxidase variants and by recombining human placental alkaline phosphates and calf intestinal alkaline phosphatase for better mutants with improved properties.