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S. L. Rosenblum, A. G. Weiden, E. L. Lewis, A. L. Ogonowsky, H. E. Chia, S. E. Barrett, M. D. Liu, and A. M. Leconte. 2017. Design and discovery of new combinations of mutant DNA polymerases and modified DNA substrates. Chembiochem 18: 816. Full Article

Chemical modifications can enhance the properties of DNA by imparting nuclease resistance or generating more diverse physical structures. However, native DNA polymerases generally cannot synthesize any length of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2’ modified DNAs; however, it is limited by the length of the products that it can synthesize. Here, we have rationally designed and characterized ten mutants of SFM19. From this library of ten mutant polymerases, we identifed enzymes with substantially improved activity relative to SFM19 for synthesis of 2’F, 2’OH, 2’OMe, and 3’OMe modified DNA as well as for reverse transcription of 2’OMe DNA. We also evaluated mutant DNA polymerases previously only tested for synthesis for 2’OMe DNA and show that they are also capable of an expanded array of modified DNA synthesis. Collectively, this work significantly expands the known combinations of modified DNA and Taq DNA polymerase mutants.

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