Patent ID: 7833759

Claim:
An in vitro method of obtaining a polynucleotide variant, comprising the steps of: a) preparing at least one first heteroduplex polynucleotide from partially complementary polynucleotide strands, said first heteroduplex polynucleotide having at least two mismatched base pairs; b) combining said first heteroduplex polynucleotide with an effective amount of purified enzymes, wherein the enzymes comprise a polymerase, a ligase and a mismatch recognizing and mismatch-directed endonuclease; c) allowing sufficient time for the percentage of complementarity between the strands of said first heteroduplex polynucleotide to increase so that variants are made that have sequences different from any of said partially complementary polynucleotide strands; d) screening or selecting for a population of variants having a desired functional property from the variants; e) denaturing at least one of said population of variants to obtain single strand polynucleotides; f) annealing said single strand polynucleotides and forming at least one second heteroduplex polynucleotide, wherein at least one strand in the second heteroduplex polynucleotide is a variant and said second heteroduplex polynucleotide has at least two mismatched base pairs; g) combining said second heteroduplex polynucleotide with an effective amount of purified enzymes, wherein the enzymes comprise a polymerase, a ligase and a mismatch recognizing and mismatch-directed endonuclease; h) allowing sufficient time for the percentage of complementarity between the strands of the second heteroduplex polynucleotide to increase so that additional variants are made that have sequences different from any of the partially complementary polynucleotide strands and different from any of the variants made in step c), and different from either strand in the second heteroduplex polynucleotide; i) screening or selecting for a population of additional variants having a desired functional property from the additional variants; and j) recovering an additional variant from the population of additional variants.