Patent ID: 6153410
Filing Date: 2000-11-28
Classification: C12N,C12Q

Abstract:
A method of evolving a polynucleotide toward acquisition of a desired property, comprising:a) contacting at least one template polynucleotide with a set of defined-sequence primers, the set of defined-sequence primers comprising a plurality of both forward and reverse primers;b) conducting a multi-cycle polynucleotide extension reaction on the at least one template polynucleotide and the set of defined-sequence primers, wherein:(i) in at least one cycle, the primers anneal to the at least one template polynucleotide and prime replication of the at least one template polynucleotide thereby generating a pool comprising overlapping fragments which are shorter in length than the at least one template polynucleotide and which overlap to span the at least one template polynucleotide; and(ii) in at least one subsequent cycle, the overlapping fragments generated in a previous cycle are denatured to single-stranded fragments, which anneal in new combinations formiing annealed fragments, whereby one strand of an annealed fragment primes replication of the other to form a further pool of overlapping fragments;whereby the multi-cyclic polynucleotide extension reaction is continued for sufficient cycles until the further pool of overlapping fragments includes variant forms of the at least one template polynucleotide; andc) screening or selecting the variant forms of the at least one template polynucleotide, or expression products thereof, for an altered or enhanced property relative to the at least one template polynucleotide or an expression product thereof.