FIGURE 3:

Polymerase engineering strategies. In order to generate polymerases with new or altered activity, the addition of new residues or mutations at residues already present are usually made, candidates for which can be identified from a variety of sources. The replacement of full domains is one engineering route that relies on the swapping or addition of domains from other enzymes to incorporate new activity or function. Like domain swapping, many protein engineering methods involve rational design based on information-transfer methods from comparison to other proteins or computational analysis, random mutagenesis followed by selection, or a combination of the two approaches. Targeted mutations are often knowledge-based and rely on previous studies for an understanding on how to achieve a desired effect. Random mutagenesis relies on making mutations in a less targeted fashion, often through error-prone PCR or use of random primers. Due to the large sequence space of enzymes, often large numbers of variants must be screened or selected to identify the desired functionality. Methods such as directed evolution and compartmentalized-self replication have been developed and used due to their increased sampling abilities.