Site-Directed Mutagenesis of Large Plasmids

A protocol combining recombination PCR and long-distance PCR is demonstrated to be highly accurate and rapid for sitedirected 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...

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Bibliographic Details
Published inBioTechniques Vol. 25; no. 6; pp. 1014 - 1019
Main Authors Nadin-Davis, Susan A, Chang, Shirley C
Format Journal Article
LanguageEnglish
Published Natick, MA Future Science Ltd 01.12.1998
Eaton
Subjects
Animals
Baculoviridae - genetics
Biological and medical sciences
Biotechnology
Blotting, Western
Cells, Cultured
DNA Primers
Fundamental and applied biological sciences. Psychology
Gene Expression Regulation, Viral
Genes, Viral
Genetic engineering
Genetic technics
Genetic Testing
Genetic Vectors
Glycoproteins - analysis
Glycoproteins - genetics
Insecta
Methods. Procedures. Technologies
Molecular Weight
Mutagenesis, Site-Directed
Plasmids - chemistry
Plasmids - genetics
Rabies virus - genetics
Recombination, Genetic
Reverse Transcriptase Polymerase Chain Reaction - methods
Site specific mutagenesis
Transformation, Genetic
Long distance
Homologous recombination
Glycoprotein
Site directed mutagenesis
Recombinant DNA
Baculovirus
Rabies virus
Virus
Polymerase chain reaction
Mononegavirales
Baculoviridae
Rhabdoviridae
Lyssavirus
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Summary:A protocol combining recombination PCR and long-distance PCR is demonstrated to be highly accurate and rapid for sitedirected 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.
ISSN:0736-6205
1940-9818
DOI:10.2144/98256st04