RANDOM-PRIMING IN-VITRO RECOMBINATION - AN EFFECTIVE TOOL FOR DIRECTED EVOLUTION

A simple and efficient method for in vitro mutagenesis and recombinati on of polynucleotide sequences is reported. The method involves primin g template polynucleotide(s) with random-sequence primers and extendin g to generate a pool of short DNA fragments which contain a controllab le level of point mutations. The fragments are reassembled during cycl es of denaturation, annealing and further enzyme-catalyzed DNA polymer ization to produce a library of full-length sequences. Screening or se lecting the expressed gene products leads to new variants with improve d functions, as demonstrated by the recombination of genes encoding di fferent thermostable subtilisins in order to obtain enzymes more stabl e than either parent.