A METHOD OF INCREASING COMPLEMENTARITY IN A HETERODUPLEX POLYNUCLEOTIDE

摘要

We describe here an in vitro method of increasing complementarity in aheteroduplex polynucleotide sequence. The method uses annealing of oppositestrands to form a polynucleotide duplex with mismatches. The heteroduplexpolynucleotide is combined with an effective amount of enzymes having strandcleavage activity, 3' to 5' exonuclease activity, and polymerase activity, andallowing sufficient time for the percentage of complementarity to be increasedwithin the heteroduplex. Not all heteroduplex polynucleotide will necessarilyhave all mismatches resolved to complementary. The resulting polynucleotide isoptionally ligated. Several variant polynucleotide result. At sites whereeither of the opposite strands has templated recoding in the other strand, theresulting percent complementarity of the heteroduplex polynucleotide sequenceis increased. The parent polynucleotides need not be cleaved into fragmentsprior to annealing heterologous strands. Therefore, no reassembly is required.