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Characterization of Modified‐DNA Polymerase Fidelity
Author(s) -
Seto Madison K.,
Barrett Susanna E.,
Weiden Aurora G.,
Leconte Aaron M.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00315
Subject(s) - dna polymerase , fidelity , polymerase , computational biology , dna , genetics , biology , computer science , telecommunications
Recently, several modified‐DNA (M‐DNA) polymerases have been identified which can synthesize long M‐DNAs. While these discoveries have enabled new applications of M‐DNA, these enzymes typically have poor fidelity, possessing error rates several orders of magnitude worse than native enzymes. However, to date, efforts to both quantify error rate, as well as experimental approaches towards understanding the origin of the poor fidelity of M‐DNA polymerases, have been limited. Here, we use a high‐throughput sequencing assay to characterize the error rate of leading M‐DNA polymerases, which showed that our enzyme, P1, has an error rate 1.6 times lower than SFM4‐6, the most frequently used Taq mutant for 2′M‐DNA synthesis. We also characterize the importance of individual mutations on fidelity, beginning to experimentally address the origins of fidelity, and have found that reverting even one mutation in a mutant yielded a fidelity that was 2.4 times greater than its parent, which will help us improve future M‐DNA polymerase rational design. Support or Funding Information National Science Foundation