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Lookup NU author(s): Dr Charles WinterhalterORCiD, Professor Heath MurrayORCiD
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Site-directed mutagenesis (or Quickchange) is a method that can be used to introduce single amino-acid substitutions in a protein of interest based on a template plasmid (Liu and Naismith, 2008). One pair of mutagenic oligonucleotides is required per mutation, and creating these must follow specific design guidelines. Therefore, generating mutant primers can be a tedious process, which becomes increasingly laborious as the number of substitutions to make rises.Here, we created a program for the automation of Quickchange primer design. The software takes a DNA sequence encoding a protein as an input and generates oligonucleotide pairs for the mutagenesis of every amino-acid in that protein within seconds. Design rules are optimised for successful PCR amplification via the Q5 DNA polymerase and based on: (i) the melting temperature (TM) of the oligonucleotide part that anneals to the template plasmid DNA, (ii) the TM corresponding to a primer pair overlapping section, (iii) the GC-content within different sections of individual primers, (iv) the presence of a GC-clamp at every oligonucleotide 3’-end, and (v) the TM difference between forward and reverse primer pairs. Settings allow to change the annealing temperature of recombinant primers according to the basic parameters of PCR reactions including final buffer salt and oligonucleotide concentrations.We applied this program to the alanine scan of the B. subtilis DnaD protein and it can be used for the systematic mutagenesis of any protein of interest.
Programmer(s): Winterhalter C, Murray H
Publication type: Software
Publication status: Published
Year: 2021
Print publication date: 30/09/2021
Publisher: Zenodo
Place Published: Geneva
URL: https://doi.org/10.5281/zenodo.5541537
DOI: 10.5281/zenodo.5541537
Notes: Is supplement to: Journal article: 10.1093/nar/gkac1060 (DOI) https://doi.org/10.1093/nar/gkac1060
