Patent ID: 7235386

Claim:
A 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 heteroduplex polynucleotide with an effective amount of purified enzymes, wherein the enzymes comprise a polymerase, a ligase and a mismatch directed endonuclease selected from the group consisting of CEL I, T4 endonuclease VII, T7 endonuclease I, and SP nuclease; c) allowing sufficient time for the percentage of complementarity between the strands of said 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; denaturing at least one of said population of variants to obtain single strand polynucleotides; f) annealing said single strand polynucleotides to form at least one second heteroduplex polynucleotide, wherein at least one strand in the second heteroduplex 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 directed endonuclease selected from the group consisting of CEL I, T4 endonuclease VII, T7 endonuclease I, and SP nuclease; h) allowing sufficient time for the percentage of complementarity between the strands of the 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 being different from either strand in the second heteroduplex; i) screening or selecting for a population of additional variants having a desired functional property from the additional variants different from any of the variants made in step c) and different from any of said partially complementary polynucleotide strands; and j) recovering an additional variant from the population of additional variants.