Site saturation mutagenesis uses primers with a degenerate codon to scramble
a single residue. Quick-quality control (QQC) is a way to determine extent
of randomisation by sequencing the pool of plasmids, measuring the peaks
and calculating the deviation from an ideal mix (Acevedo-Rocha *et al.*, 2015 ).

This tool performs automatically the peak measurements, Q-value calculations and expected amino acid proportions. In the case of sequences mutated with multiple codons a rough estimate of the contributions of each is obtained by optimining the function:

\(\min_{\boldsymbol{x} \in{\Bbb{N}}} \sum\limits_{i=1; j=1}^{4; 3} \left|{\sum\limits_{k=1}^{n} d_k x_{ijk} h_{ijk} - m_{ij}}\right|\)

Where **x** is a scaling factor (4x3xN tensor) of the deviation
from expected, **m**
is the empirical proportions of bases (4x3
matrix), **d** is the proportions of the primers (N dimension vector)
and **h** is the ideal proportionl of bases in each primer (4x3xN
tensor).

This approach therefore tries to balance the primers so to minimise the deviation from the ideal primer mix. The usage of the ideal primer mix is unfortunate, but is the only way to tackle the problem of deconvoluting the primers.

Upstream seq.

Scheme

Direction