Research@StAndrews
 
The University of St Andrews

Research@StAndrews:FullText >
University of St Andrews Research >
University of St Andrews Research >
University of St Andrews Research >

Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/2456
This item has been viewed 5 times in the last year. View Statistics

Files in This Item:

File Description SizeFormat
LiuNaismith2008BMCBiotechnology8_91.pdf757.11 kBAdobe PDFView/Open
Title: An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol
Authors: Liu, Huanting
Naismith, James Henderson
Keywords: Polymerase-chain-reaction
Overlap extension
Primer extension
DNA
PCR
Hybridization
Mutations
Selection
Cloning
QD Chemistry
Issue Date: 4-Dec-2008
Citation: Liu , H & Naismith , J H 2008 , ' An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol ' BMC Biotechnology , vol 8 , 91 .
Abstract: Background: Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable. Results: We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange (TM) site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis. Conclusion: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange (TM). Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange T) but increased the overall success rates.
Version: Publisher PDF
Status: Peer reviewed
URI: http://hdl.handle.net/10023/2456
DOI: http://dx.doi.org/10.1186/1472-6750-8-91
ISSN: 1472-6750
Type: Journal article
Rights: © 2008 Liu and Naismith; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Appears in Collections:University of St Andrews Research
Chemistry Research
Biomedical Sciences Research Complex (BSRC) Research



This item is protected by original copyright

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

 

DSpace Software Copyright © 2002-2012  Duraspace - Feedback
For help contact: Digital-Repository@st-andrews.ac.uk | Copyright for this page belongs to St Andrews University Library | Terms and Conditions (Cookies)