Site directed Mutagenesis-Primer Design

We received proteorhodopsin sequence from Kwang-Hwan "Kevin" Jung, Ph.D., Associate Professor, Department of Life Science and Institute of Biological Interfaces, Sogang University with three restriction sites EcoRI, SpeI and PstI. Hence the plasmid had to be mutated for iGEM compatibility. Hence we had to design primers for site directed mutagenesis at each of these sites. So we have come up with a Primer design tool for SDM using following criterias:

1. Mutations in the middle of primer
2. No 3' Breathing should be ensured by keeping G or C at the end
3. The Tm and GC content are taken as user inputs.

The novelty here is that the complete protein sequence and all the sites at which a specific mutation is required can be given at a time. This would generate the best possible primers required also accounting for distance between the mutations hence optimizing the number of primers.

Click here to download the perl code

Fig(1): The Command Prompt, while using the Primer design tool.

Fig(2): The Primer designs obtained using the toolbox for various mutations

Fig(3): The Primer designs obtained using the toolbox for various mutations (Common Primer designed for mutations in close proximity