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Deepscan

Primer design for deep mutational scanning

Description

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 ).

Input

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.
To give a sequence with a start and end position. A variant of this is to give the starting/end position as a short sequence to match as opposed to a number.

Upstream
Sequence

Sequence

Downstream
Sequence

 
First base

Last base

Mutation

Enzyme

Target Tm
°C

GC clamp bonus
°C

Method

Overlap
nt

Length