Directed evolution of DNA polymerases.

摘要

To achieve the long-term goal of the Benner research group to create a synthetic biology based on an Artificially Expanded Genetic Information System (AEGIS), polymerases that are able to incorporate non-standard bases (NSBs) into DNA must be identified. In this dissertation, a polymerase from Thermus aquaticus (Taq Polymerase) was identified that was able to incorporate non-standard nucleotide analogs that contain a C-glycosidic linkage. This activity was limited, meaning that the polymerase needed modification to support this goal. Further, we asked whether sequential C-glycosides destabilized the duplex and altered its structure, to better understand whether a synthetic biology based on C-glycoside nucleotides was possible.; To this end, two libraries of polymerases were created to identify mutations necessary to alter the polymerases' ability to withstand high temperatures. One library was created by the random mutagenesis of the taq gene, the other was rationally designed based on previous studies. Seventy-four mutants from each library were screened for their ability to generate a full-length polymerase chain reaction (PCR) product using standard nucleoside triphosphates at various temperatures; the library of random mutants contained more thermostable polymerases than the library obtained by rational design. Water-in-oil emulsions were then tested to determine whether these, as artificial cells, might deliver thermostable polymerase variants from those used in the screen. This identified difficulties in tools used to analyze the output of the library, suggesting solutions that will guide future work. We also tested the individual components of the rationally designed library for their ability to incorporate C-glycoside triphosphates in a PCR. Structural studies with synthetic DNA containing multiple, consecutive C-glycosides showed no change in conformation, at least not one that is detectable by circular dichroism.; These results represent a step towards the goal of creating an AEGIS-based synthetic biology, an artificial chemical system that mimics emergent biological behaviors such as replication, evolution, and adaptation. In addition, the mutant polymerases created in these experiments are an inventory of polymerases useful in biotechnology, possibly allowing the development of new, as well as improving on existing, clinical diagnostic techniques and helping to facilitate a better understanding of polymerase-DNA interactions.