RECOMBINATION OF POLYNUCLEOTIDE SEQUENCES USING RANDOM OR DEFINED PRIMERS

摘要

PURPOSE: A recombining method is provided to form a pool of short DNA fragments by priming template poly nucleotide with random-sequences or defined-sequence primers. CONSTITUTION: A method for in vitro mutagenesis and recombination of poly nucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template poly nucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturalization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template poly nucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.