Library construction and evaluation for site saturation mutagenesis

Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy

“…The wild-type codon sources are mainly from parent template that survived the Dpn I digestion and the wild-type codon present in the NNK primer mixture. Therefore, although it has been shown that in the case of an enoate reductase this method works better than earlier approaches (Sullivan et al 2013), it performs poorly when turning to the more challenging difficult-to-randomize gene P450-BM3.…”

“…This “pooled” plasmid sample was then sequenced, and the resulting capillary electropherograms were assessed (Figs. S2–S6) and the Q pool values were calculated to evaluate the degeneracy based on the reported method (Sullivan et al 2013). Ninety-six individual colonies were picked from another plate and inoculated into 400 μL of LB containing 50 μg/mL kanamycin in a 2-mL 96-well plate.…”

“…The PCR reaction is a linear, rather than an exponential amplification process due to the completely overlapped primers (Xia et al 2015). Since the products from the first cycle cannot serve as templates for subsequent rounds, the PCR amplicon yield is rather low, which means that it is very difficult to obtain the desired number of colonies after transformation (Sullivan et al 2013). To overcome this problem, different approaches have been developed by using partially overlapped (Zheng et al 2004) or even non-overlapping primers (Kirsch and Joly 1998), where the resulting amplicon is used as a megaprimer (Sarkar and Sommer 1990; Miyazaki and Takenouchi 2002), thereby completing the plasmid amplification in a second stage PCR.…”

Enhancing the heat stability and kinetic parameters of the maize endosperm ADP-glucose pyrophosphorylase using iterative saturation mutagenesis

“…The relative peak heights for each base (which correspond to the relative nucleotide compositions of the mixed plasmid population) were calculated for each of the targeted bases at the appropriate locations on the chromatograms. These were used to calculate values for Q codon as described previously and used to estimate the number of amino acids likely to be present at each randomized position [21]. A Q codon value of 1.0 represents perfect randomization while 0.0 indicates no sequence diversity.…”

“…We constructed nine random replacement libraries for the maize AGPase large subunit using PCR-based methodology [21] that provided high-quality libraries with little contamination by the parent sequence and undetectable levels of concatemeric primer inserts. Both defects would otherwise dilute the library and add to the screening burden.…”