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Compartmentalized Self-Replication for Evolution of a DNA Polymerase.

Zhanar Abil1, Andrew D Ellington1,2

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|July 25, 2018
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Summary

Compartmentalized self-replication (CSR) enables DNA polymerase engineering by selecting active variants. This emulsion PCR method amplifies genes encoding high-performing polymerases for improved enzyme activity.

Keywords:
CSRDNA polymeraseDirected evolutioncompartmentalized-self replicationemulsion PCRin vitro compartmentalizationselection

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Enzyme Engineering

Background:

  • Compartmentalized self-replication (CSR) is an emulsion PCR-based technique.
  • It facilitates the selection and engineering of DNA polymerases.
  • Existing methods require improvements for efficiency and substrate versatility.

Purpose of the Study:

  • To provide a detailed protocol for DNA polymerase selection using CSR.
  • To demonstrate the application of CSR for engineering polymerase activity.
  • To highlight CSR's utility in selecting variants with novel functionalities, such as reverse transcriptase activity.

Main Methods:

  • Emulsification of E. coli host cells expressing DNA polymerase libraries.
  • In-droplet PCR amplification of genes encoding active polymerase variants.
  • Iterative rounds of selection and amplification for enzyme engineering.

Main Results:

  • Successful selection of DNA polymerase variants with enhanced activity.
  • Demonstration of CSR for selecting reverse transcriptase activity from a library of Thermococcus kodakaraensis (KOD) DNA polymerase variants.
  • Exponential amplification of genes encoding superior polymerase variants.

Conclusions:

  • CSR is a powerful and versatile method for DNA polymerase engineering.
  • The protocol enables selection under diverse conditions, including non-standard substrates.
  • CSR facilitates the directed evolution of enzymes for improved performance and novel functions.