Chemical Biology of DNA Polymerases : From Selectivity to New Functions

Abstract

DNA polymerases are involved in all DNA synthesis occurring in nature. Furthermore, DNA polymerases are the workhorses in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing and nucleic acids based diagnostics.In order to identify DNA polymerase mutants with altered properties, we set up an efficient high through put setup to rapidly screen libraries of DNA polymerase mutants in automated parallel fashion and identified entities with significantly increased selectivity. Furthermore, our results indicate a rational to generally increase DNA polymerase selectivity as we demonstrate for several enzymes from different DNA polymerase families.Additionally, we show that the generation of a new DNA polymerase function is achievable through iterative screening of small libraries of DNA polymerase derived by randomization of the respective genes. We demonstrate that the identified mutants find immediate applications and provide the basis for the development of new means for diagnostic technologies.