Deepscan generates a list of partially overlapping QuikChange-based primers
aimed at constructing mutability landscapes using PCR. It iterates across
a DNA sequence, codon by codon and generates a primer pair that employs
the partially overlapping primer pair strategy (Xia et al., 2015 ).
Namely, the primers in a pair will have an user-defined overlap length
( e.g. 22 bp) centred around the codon to mutate and will have a 3’
overhand long enough to allow the region beyond the mutagenized codon to
anneal with the template above a given melting temperature, while taking
into account terminal GC clamp. The traditional Quikchange protocol (Agilent)
works poorly for codon mutants.
The serverside calculations use a Python 3 script obtainable from: mutational scanning ).
To make primers at the ends of the coding sequence, the neighbouring noncoding sequence are required. Two options are available:To give the upstream and downstream sequence separately.