Question

Assume there is a plasmid, pAGG1 that carries two genes, geneA encoding the antibiotic resistance gene to select the plasmid and geneX which is your gene of interest. Each of the two genes is about 0.6kb encoding proteins of about 200 amino acids.

Question:

Discuss what limitations there are in mutagenizing the entire plasmid to saturate geneX, when would you select a whole gene method to mutagenize geneX versus a site directed method?

Answer 1

Saturation mutagenesis involves the use of randomised codons (NNK or NNS) in the primer for PCR amplification of a plasmid. This allows the production of all possible amino acids at the target codon and thus a large number of mutants can be generated very easily. Sequence saturation mutagenesis allows the simultaneous incorporation of multiple mutations across the gene of interest via the use of modified nucleotides during the PCR. A major limitation of saturation mutagenesis is the loss of diversity or functionality due to the use of degenerate primers. Furthermore, extensive screening is required to detect the mutants with desirable traits when the number of degenerate sites is high. Another issue is the possibility of random incorporation of termination codons.

The technique for a simple PCR based site directed mutagenesis (SDM) involves the use of primers with a mutation. The mutated primer is elongated via PCR using the complementary plasmid strand as template, and a nicked circular DNA is produced. The original plasmid is digested using restriction enzymes and the new circular DNA is used as the template in subsequent PCR steps. Although quite simple, the technique allows only linear amplification and not an exponential increase in the amplicon like normal PCR products. Also each type of mutation will require a dedicated primer. Thus, in order to generate different mutants, separate reactions with specific primers will have to be set. Saturation mutagenesis is adopted to overcome these limitations.