MOLECULAR EVOLUTION BY STAGGERED EXTENSION PROCESS (STEP) IN-VITRO RECOMBINATION

We have developed a simple and efficient method for in vitro mutagenes is and recombination of polynucleotide sequences. The staggered extens ion process (StEP) consists of priming the template sequence(s) follow ed by repeated cycles of denaturation and extremely abbreviated anneal ing/polymerase-catalyzed extension. In each cycle the growing fragment s anneal to different templates based on sequence complementarity and extend further, This is repeated until full-length sequences form, Due to template switching, most of the polynucleotides contain sequence i nformation from different parental sequences, The method is demonstrat ed by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers, To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabili zed subtilisin E variants identified during a single round of error-pr one PCR mutagenesis and screening. Screening the StEP-recombined libra ry yielded an enzyme whose half-life at 65 degrees C is 50 times that of wild-type subtilisin E.