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Directed evolution of custom polymerases using droplet microfluidics.

Derek Vallejo1, Ali Nikoomanzar1, John C Chaput1

  • 1Departments of Pharmaceutical Sciences, Chemistry, Molecular Biology, and Biochemistry, University of California, Irvine, CA, United States.

Methods in Enzymology
|September 18, 2020
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Summary

This study introduces a novel microfluidic method for directed evolution of DNA polymerases. The technique enables rapid screening and isolation of engineered polymerases with desired functions for biotechnology applications.

Keywords:
Droplet sortingMicrofluidicsPolymerase

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biochemistry

Background:

  • DNA polymerases are essential for biotechnology but desired variants are often unavailable.
  • Directed evolution offers a method to engineer polymerases for specific unnatural substrates.

Purpose of the Study:

  • To develop a microfluidic-based ultrahigh throughput method for evolving new DNA polymerase functions.
  • To enable the creation of engineered polymerases with enhanced recognition of unnatural substrates.

Main Methods:

  • Utilized a microfluidic system for ultrahigh throughput sorting of fluorescent water-in-oil microdroplets.
  • Involved E. coli expression of diverse polymerase variants, droplet encapsulation, bacterial lysis, fluorescence-based activity assay, and fluorescence-activated droplet sorting (FADS).
  • Employed plasmid recovery and DNA sequencing to identify functional variants.

Main Results:

  • Successfully demonstrated a microfluidic workflow for directed evolution of DNA polymerases.
  • Identified engineered polymerase variants with desired activities through fluorescence-based sorting.
  • Established a method amenable to various unnatural nucleic acids and polymerase functions.

Conclusions:

  • The developed microfluidic method provides an efficient platform for engineering novel DNA polymerase functions.
  • This technique significantly advances the ability to create custom polymerases for diverse biotechnological applications.
  • The approach is versatile and applicable to DNA-templated synthesis, reverse transcription, and replication.