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Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases.

Mirkó Palla^1,2, Sukanya Punthambaker^1,2, Benjamin Stranges¹

¹Harvard Medical School, Department of Genetics, Boston, Massachusetts 02115, United States.

|December 28, 2020

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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.

Keywords:

kinetic characterization multiplex barcoding nanopore sequencing polymerase screening single-molecule detection

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Last Updated: Nov 24, 2025

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

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Published on: April 4, 2025

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

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Published on: May 6, 2020

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

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Published on: June 3, 2019

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.