Evolution of a Thermophilic Strand-Displacing Polymerase Using High-Temperature Isothermal Compartmentalized Self-Replication

John N. Milligan, Raghav Shroff, Daniel J. Garry, Andrew D. Ellington

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions, where the lack of thermal cycling reduces equipment needs and improves the time to answer, especially for point-of-care applications. In order to improve the function of strand-displacing polymerases, we have developed an emulsion-based directed evolution scheme, high-temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand-displacing polymerase variant that performs well in loop-mediated isothermal amplification and rolling circle amplification, even after thermal challenges of up to 95 °C that lead to better primer annealing. The new enzyme (v5.9) is also capable of a variety of new reactions, including isothermal hyperbranched rolling circle amplification. The HTI-CSR method should now prove useful for evolving additional beneficial phenotypes in strand-displacing polymerases.

Original languageEnglish (US)
Pages (from-to)4607-4619
Number of pages13
Issue number31
StatePublished - Aug 7 2018

Bibliographical note

Funding Information:
This work was supported by the Air Force Office of Scientific Research (FA9550-14-1-0089 to A.E.), the National Science Foundation (1541244 to A.E.), and the National Institutes of Health (1T32LM012414-01A1 to R.S.). Notes The authors declare no competing financial interest.

Publisher Copyright:
© 2018 American Chemical Society.


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