Patent ID: 8883453

Claim:
A method of codon-specific mutagenesis, the method comprising: providing a first plasmid comprising a target open reading frame, a first reading-frame selectable marker, and an origin of replication, wherein the first plasmid does not contain a BsgI restriction site; providing a linear Mu transposon comprising a double stranded nucleic acid with first and second ends, second and third selectable markers, a mutant codon proximal the first end, wherein the first and second ends comprise overhanging DNA sequence (sticky ends), and wherein at least the second selectable marker is in the same translational reading frame as the mutant codon; reacting the Mu transposon and first plasmid in the presence of MuA transposase to cause integration of the transposon into the plasmid at an insertion site with accompanying removal of the sticky ends and duplication of a five base pair sequence of first plasmid at the insertion site to form a second plasmid; performing an inverse-polymerase chain reaction (inverse-PCR) employing the second plasmid as a template and first and second oligonucleotide primers, wherein each primer comprises an overhanging nucleic acid sequence and a template binding sequence, wherein the overhanging nucleic acid sequence comprises a BsgI restriction site, wherein the inverse PCR produces a second linear double-stranded nucleic acid; digesting the second linear double-stranded nucleic acid with BsgI restriction enzyme to form a third linear double-stranded nucleic acid, wherein the third double-stranded nucleic acid comprises the mutant replacement codon, the first reading-frame selectable marker, and first and second ends with overhanging nucleic acid sequence; repairing the third double-stranded nucleic acid with a proofreading polymerase to form a fourth double-stranded nucleic acid with blunt ends; and ligating the fourth double-stranded nucleic acid sequence intramolecularly to form a third plasmid, wherein the third plasmid encodes a mutant polypeptide.