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Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases
Mirkó Palla1,2, Sukanya Punthambaker1,2, Benjamin Stranges1
1Harvard Medical School, Department of Genetics, Boston, Massachusetts 02115, United States.
ACS Nano
|December 28, 2020
Summary
Researchers developed a rapid, single-molecule method to screen DNA polymerase variants using nanopore sequencing. This technique allows for parallel analysis of many variants, identifying enzymes with desirable biotechnological properties.
Area of Science:
- Biotechnology
- Molecular Biology
- Biophysics
Background:
- DNA polymerases are crucial for genetic information replication and have applications in biotechnology.
- Previous work established a single-molecule DNA sequencing platform using DNA polymerase and nanopore technology.
Purpose of the Study:
- To develop a multiplexed, single-molecule screening method for DNA polymerase variants.
- To identify novel polymerase variants with altered physical properties for biotechnological applications.
Main Methods:
- Complexing barcoded DNA strands with DNA polymerase variants.
- Utilizing nanopore sequencing to analyze barcoded DNA, revealing barcode identity and polymerase kinetic properties.
- Developing a classification algorithm to differentiate kinetic characteristics of polymerase mutants.
Main Results:
- Demonstrated a single-molecule method for rapid, multiplexed screening of DNA polymerase variants.
- Successfully screened a library of approximately 100 polymerase variants.
- Identified polymerase variants with potential biotechnological applications.
Conclusions:
- The developed method enables parallel investigation of numerous polymerase variants on a single nanopore array.
- This approach facilitates the identification of DNA polymerases with specific, altered physical properties.
- The screening method is broadly applicable for discovering novel polymerases for various biotechnological uses.
