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One‐Step site‐directed mutagenesis of ATM cDNA in large (20kb) plasmid constructs
Author(s) -
Scott Shaun P.,
Teh Alison,
Peng Cheng,
Lavin Martin F.
Publication year - 2002
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.9068
Subject(s) - biology , genetics , mutagenesis , complementary dna , site directed mutagenesis , plasmid , computational biology , microbiology and biotechnology , mutation , dna , gene , mutant
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. © 2002 Wiley‐Liss, Inc.