Rationally Designed Mutant Libraries
Custom mutant library construction using gene synthesis is most effective for characterizing small numbers of variants (10’s), as the costs increase rapidly when building libraries to comprehensively uncover critical positions and identify improved variants across a complete domain.
A new high-throughput site-directed mutagenesis method (Figure 1) dramatically reduces the cost and limitations of custom mutant library synthesis, while overcoming the screening burden posed by site-directed mutagenesis with degenerate (mixed base) codon primers. This approach combines the high fidelity of massively parallel oligo synthesis with the ease of use and efficiency of QuikChange site-directed mutagenesis. This oligo library method enables rationalized single amino acid (e.g., Alanine) scanning, codon saturation scanning and targeted combinatorial mutagenesis, providing structural and functional linkage while identifying improved variants. The rationally designed library minimizes the number of clones to screen, without the prohibitive expense of synthesizing the entire gene.
This new approach begins with the in silico design of as many as tens of thousands of user-defined sequences using a dedicated web-based software (Figure 1). Every codon within a 20–70 amino acid region can be precisely targeted with a single oligo set. Multiple oligo sets can be designed to mutate continuous or discrete sites, including other regions of the same or different proteins, in parallel reactions. This rational design approach eliminates the codon redundancy and bias, as well as the non-coding, wild type and non-target mutations observed when using degenerate oligos and error-prone (EP)-PCR.
Once the oligo sets are produced and received by the researcher, they are amplified via PCR, purified, and incorporated into an appropriate plasmid vector (containing the wild-type GOI and propagated in an X strain) using linear amplification and high-fidelity Pfu DNA polymerase to minimize the frequency of unintended secondary mutations outside the primer-binding region (Figure 1). The methylated and hemi-methylated parental plasmid is then digested with Dpn I enzyme, and the mutant plasmids are transformed into supercompetent cells developed for this application.