Patent ID: 8383346

Claim:
A method of synthesizing a plurality of polynucleotide variants each having at least one defined nucleotide difference relative to a single starting reference polynucleotide sequence, the method comprising: (a) separately amplifying a single starting reference polynucleotide template with each of a plurality of pairs of forward and reverse mutagenic primers, wherein the plurality of pairs of forward and reverse mutagenic primers comprises the plurality of defined nucleotide differences and wherein each pair generates an amplicon comprising a sequence capable of binding to an adjacent overlapping sequence of at least one other amplicon, wherein the plurality of pairs of forward and reverse primers comprise at least one degenerate primer, and wherein said amplifying is conducted in one round of simultaneous amplification utilizing said plurality of pairs of forward and reverse mutagenic primers to generate said amplicons; (b) purifying said amplicons to provide purified amplicons; (c) separately assembling a plurality of sets of said purified amplicons, wherein each set comprises amplicons having adjacent overlapping sequences capable of binding to form the full length of the reference polynucleotide sequence, wherein the sequence comprises mutations introduced by said mutagenic primers; (d) replicating the plurality of sets of assembled amplicons, thereby synthesizing a plurality of polynucleotide variants; and (e) combining the plurality of polynucleotide variants.