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Protocol

The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection

1
Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK
2
Clatterbridge Cancer Centre NHS Foundation Trust, Clatterbridge Road, Bebington CH63 4JY, UK
*
Author to whom correspondence should be addressed.
Methods Protoc. 2021, 4(1), 14; https://doi.org/10.3390/mps4010014
Received: 25 January 2021 / Revised: 12 February 2021 / Accepted: 13 February 2021 / Published: 16 February 2021
(This article belongs to the Section Molecular and Cellular Biology)
The comet assay is a versatile, simple, and sensitive gel electrophoresis–based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1–2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD. View Full-Text
Keywords: comet assay; DNA damage; complex DNA damage; DNA repair; ionising radiation; protons comet assay; DNA damage; complex DNA damage; DNA repair; ionising radiation; protons
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MDPI and ACS Style

Fabbrizi, M.R.; Hughes, J.R.; Parsons, J.L. The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection. Methods Protoc. 2021, 4, 14. https://doi.org/10.3390/mps4010014

AMA Style

Fabbrizi MR, Hughes JR, Parsons JL. The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection. Methods and Protocols. 2021; 4(1):14. https://doi.org/10.3390/mps4010014

Chicago/Turabian Style

Fabbrizi, Maria R., Jonathan R. Hughes, and Jason L. Parsons 2021. "The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection" Methods and Protocols 4, no. 1: 14. https://doi.org/10.3390/mps4010014

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