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Related Experiment Video

Updated: Apr 27, 2026

Studying DNA Looping by Single-Molecule FRET

Studying DNA Looping by Single-Molecule FRET

Published on: June 28, 2014

Studying DNA looping by single-molecule FRET.

Tung T Le¹, Harold D Kim²

¹School of Physics, Georgia Institute of Technology.

Journal of Visualized Experiments : Jove

|July 8, 2014

Summary

This study introduces a ligase-free method using Fluorescence Resonance Energy Transfer (FRET) to measure DNA looping kinetics. The new approach accurately quanties DNA looping probability, or J factor, revealing sensitivity to DNA

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Last Updated: Apr 27, 2026

Studying DNA Looping by Single-Molecule FRET

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Published on: June 28, 2014

Design and Synthesis of a Reconfigurable DNA Accordion Rack

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Published on: August 15, 2018

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

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Published on: September 27, 2024

Area of Science:

Molecular Biology
Biophysics
Biochemistry

Background:

DNA bending is crucial for biological processes like DNA-protein interactions and packaging.
Traditional DNA cyclization assays using DNA ligase are influenced by factors unrelated to looping, such as DNA structure and enzyme binding.
A need exists for methods to measure DNA looping kinetics without ligase interference.

Purpose of the Study:

To develop and validate a novel method for measuring double-stranded DNA (dsDNA) looping kinetics without the use of DNA ligase.
To quantify the looping probability density, or J factor, of dsDNA molecules.
To assess the sensitivity of the J factor to the intrinsic curvature of dsDNA.

Main Methods:

Developed a ligase-free assay utilizing Fluorescence Resonance Energy Transfer (FRET) to detect transient DNA loop formation.
Constructed dsDNA molecules using a Polymerase Chain Reaction (PCR)-based protocol incorporating a FRET pair and a biotin linker.
Extracted the J factor from measured looping and annealing rates between sticky ends.

Main Results:

Successfully measured dsDNA looping kinetics without ligase.
The J factor was extracted from looping and annealing rates.
Demonstrated that the J factor is sensitive to the intrinsic curvature of dsDNA, as shown by testing two dsDNAs with different curvatures.

Conclusions:

The developed FRET-based method provides a ligase-free approach to study DNA looping kinetics.
This method allows for accurate measurement of the J factor, reflecting DNA looping probability.
The J factor's sensitivity to intrinsic DNA shape opens new avenues for studying DNA mechanics and protein-DNA interactions.