Related Experiment Video
Updated: May 13, 2026
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites
Abhijit Karnik1, Rucha Karnik, Christopher Grefen
1Department of Computer Science, University of Bristol, Bristol, UK.
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.
