Method for generating recombinant DNA library using unidirectional single-stranded DNA fragments

The present invention relates to a method for producing a recombinant polynucleotides comprising the steps of generating a pool of unidirectional single-stranded polynucleotide fragments from two or more homologous double-stranded polynucleotides, conducting a polymerization process comprising multi-cyclic extension reactions using the unidirectional single-stranded polynucleotide fragments as templates and specific oligonucleotides as primers to obtain recombinant polynucleotides, and conducting a polymerase chain reaction using at least one primer to amplify the recombinant polynucleotides; and a method for constructing a recombinant DNA library comprising the steps of inserting the recombinant polynucleotide prepared by the above method into a vector and transforming an expression cell with said vector containing the recombinant polynucleotide to obtain a plurality of mutant clones. The method of the present invention can be used for in vitro recombination of homologous polynucleotides and the directed molecular evolution.