One-step site-directed mutagenesis of ATM cDNA in large (20kb) plasmid constructs.
In vitro mutagenesis of large genes has proven to be difficult for a number of reasons, including the number of steps involved and the instability of large inserts. We describe here a single-step PCR method to introduce mutations into an ataxia-telangiectasia mutated (ATM) gene cDNA construct (20 kb). This involved several modifications of the Stratagene QuikChange Site-Directed Mutagenesis Kit. Four sites implicated in the function of ATM were mutagenised in a 20 kb plasmid, improving upon existing methods capable of mutagenesis in DNA constructs up to 13 kb, while maintaining a high efficiency of mutagenesis (85-100%). This approach makes it possible to introduce multiple mutations into large cDNA for structural/functional studies.
D001260:Ataxia Telangiectasia / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary / Q000235:genetics*; D004268:DNA-Binding Proteins; D005821:Genetic Techniques; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D025521:Tumor Suppressor Proteins
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011933:Reagent Kits, Diagnostic; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human