One-Step site-directed mutagenesis of ATM cDNA in large (20kb) plasmid constructs

Scott, Shaun P., Teh, Alison, Peng, Cheng and Lavin, Martin F. (2002) One-Step site-directed mutagenesis of ATM cDNA in large (20kb) plasmid constructs. Human Mutation, 20 4: 323-326. doi:10.1002/humu.9068


Author Scott, Shaun P.
Teh, Alison
Peng, Cheng
Lavin, Martin F.
Title One-Step site-directed mutagenesis of ATM cDNA in large (20kb) plasmid constructs
Journal name Human Mutation   Check publisher's open access policy
ISSN 1098-1004
1059-7794
Publication date 2002
Sub-type Article (original research)
DOI 10.1002/humu.9068
Volume 20
Issue 4
Start page 323
End page 326
Total pages 4
Place of publication New York ; Brisbane
Publisher Wiley-Liss
Language eng
Subject 0601 Biochemistry and Cell Biology
Abstract 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.
Keyword ATM
Ataxia-telangiectasia mutated
Site-Directed Mutagenesis
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Medicine Publications
 
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Created: Tue, 24 Mar 2009, 11:02:19 EST by Ms Sarada Rao on behalf of UQ Centre for Clinical Research