Finding That Molecule in a Haystack
In some sense, directed evolution is a numbers game whereby the possibility to find the desired mutant is proportional to the library size. Thus, creating a high-quality genetic library is a critical step for a successful directed evolution campaign.
Methods for the creation of genetic diversity can be broadly classified into three categories * random mutagenesis * focused mutagenesis * and DNA recombination.
Error-prone PCR primarily represents random mutagenesis. It is frequently used whenever there is not enough knowledge about the target molecule. Its simplicity also favours its wide use and makes it the first choice mutagenesis method in many labs. In the focused mutagenesis, a sweat of techniques was developed to insert mutations in the selected position(s) in the gene of interest. It represents a more 'surgical' approach, and it is utilised whenever an active site of the protein or a particular binding interface is to be evolved while keeping the rest of the protein fold native. DNA recombination combines both methods' benefits as it is more efficient than error-prone PCR, and it, in principle, can lead to even mutagenesis of the whole gene of interest.