SDM-Assist software to design site-directed mutagenesis primers introducing “silent” restriction sites

Karnik, A., Karnik, R. and Grefen, C. (2013) SDM-Assist software to design site-directed mutagenesis primers introducing “silent” restriction sites. BMC Bioinformatics, 14, 105. (doi:10.1186/1471-2105-14-105) (PMID:23522286) (PMCID:PMC3644487)

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Abstract

Background: Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. Results: We have developed a program – ‘SDM-Assist’ which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of ‘mutated clones’ by a simple restriction digest. Conclusions: The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Grefen, Dr Christopher and Karnik, Dr Rucha
Authors: Karnik, A., Karnik, R., and Grefen, C.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:BMC Bioinformatics
Publisher:BioMed Central
ISSN:1471-2105
ISSN (Online):1471-2105
Copyright Holders:Copyright © 2013 Karnik et al.
First Published:FIrst published in BMC Bioinformatics 14: 105
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
537651Regulation of membrane fusion by a novel Sec1/Munc18-associated proteinMichael BlattBiotechnology and Biological Sciences Research Council (BBSRC)BB/H024867/1RI MOLECULAR CELL & SYSTEMS BIOLOGY