Site-directed mutagenesis of large plasmids.
A protocol combining recombination PCR and long-distance PCR is demonstrated to be highly accurate and rapid for site-directed mutagenesis of large (> 10 kb) plasmids. Application of this protocol to the generation of mutant rabies virus glycoproteins expressed by the baculovirus/insect cell system illustrates the usefulness of this approach in facilitating structure-function relationships in this important eukaryotic expression system.
Nadin-Davis
S A
SA
Animal Diseases Research Institute, Canadian Food Inspection Agency, Nepean, Ont., Canada.
Chang
S C
SC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005820:Genetic Testing; D005822:Genetic Vectors; D006023:Glycoproteins / Q000032:analysis / Q000235:genetics; D007313:Insecta; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D011820:Rabies virus / Q000235:genetics; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014170:Transformation, Genetic
D017931:DNA Primers; D006023:Glycoproteins