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

Fabbrizi, Maria Rita ORCID: 0000-0002-5156-1575, Hughes, Jonathan R and Parsons, Jason L ORCID: 0000-0002-5052-1125 (2021) The Enzyme-Modified Comet (EMNC) Assay for Complex DNA Damage Detection. Methods and Protocols, 4 (1). p. 14.

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Abstract

<jats:p>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.</jats:p>

Item Type:	Article
Uncontrolled Keywords:	comet assay, DNA damage, complex DNA damage, DNA repair, ionising radiation, protons
Depositing User:	Symplectic Admin
Date Deposited:	24 Feb 2021 15:30
Last Modified:	18 Jan 2023 22:58
DOI:	10.3390/mps4010014
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3116025