Compositions for making random codon-mutant libraries and uses thereof

A method for making a plurality of variant nucleic acid molecules, comprising

• (a) amplifying a reference nucleic acid molecule template with a plurality of forward mutagenic oligonucleotides to produce a first plurality of forward mutagenic fragments and separately amplifying the reference nucleic acid molecule template with a plurality of reverse mutagenic oligonucleotides to produce a first plurality of reverse mutagenic fragments, wherein the reference nucleic acid molecule template comprises a plurality of codons that encode a reference polypeptide and wherein each of the plurality of forward and reverse mutagenic oligonucleotides comprise a codon randomized at one to three nucleotides and the oligonucleotides comprise from nine to 100 nucleotides, and wherein the randomized codons correspond to all codons of the reference nucleic acid molecule template except the initiation codon;
• (b) purifying the first plurality of forward and reverse mutagenic fragments and mixing the purified first plurality of forward and reverse mutagenic fragments to produce a first mixed mutagenic nucleic acid molecule fragment template composition;
• and
• (c) joining the mixed nucleic acid molecule fragments of the first mixed mutagenic nucleic acid molecule fragment template composition by amplifying in a single reaction with the plurality of forward and reverse mutagenic oligonucleotides from step (a) to produce a first plurality of joined mutagenic nucleic acid molecules;
• thereby introducing codon variants at random locations at a controlled mutation rate across the length of the reference nucleic acid molecule template.