Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Mohamed A. Sobhy; Amer Brali&#263;; Vlad-Stefan Raducanu; Muhammad Tehseen; Yujing Ouyang; Masateru Takahashi; Fahad Rashid; Manal  S. Zaher; Samir M. Hamdan

doi:10.3791/60045

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Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

JoVE Journal
Genetyka

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JoVE Journal Genetyka

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

9,322 Views

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11:27 min

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September 18, 2019

DOI:

10.3791/60045-v

DOI:

10.3791/60045-v

•

11:27 min

September 18, 2019

•

38 Views

Mohamed A. Sobhy*¹, Amer Bralić*¹, Vlad-Stefan Raducanu¹, Muhammad Tehseen¹, Yujing Ouyang¹, Masateru Takahashi¹, Fahad Rashid¹, Manal S. Zaher¹, Samir M. Hamdan¹

¹Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division (BESE),King Abdullah University of Science and Technology (KAUST)

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Podsumowanie

Presented here is a protocol for performing single-molecule Förster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.