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.