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JoVE Journal

Biochemistry

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Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

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09:53 min

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February 7th, 2021

February 7th, 2021

•
1,730 Views

0:00

Introduction

0:31

Preparation of Quantum-Dot-Labeled DNA Substrate Materials

3:38

Microfluidic Device Construction

5:58

Surface Tethering of Quantum-Dot-Labeled DNA Substrate

7:07

REase-Mediated DNA Cleavage

7:51

Results: Single-Molecule TIRF Experiment for Parallelized Observations of DNA Cleavage Events

9:32

Conclusion

Transcrição

With this protocol, we can directly observe site-specific DNA cleavage by restriction endonucleases at the single molecule level. Our multiplexed approach allows us to measure the time it takes for hundreds of individual REases to complete the cat

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Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

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