The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection.
The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection.
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DOI：
10.3390/mps4010014
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发表时间：
2021-02-16
影响因子：
2.4
通讯作者：
Parsons JL
中科院分区：
文献类型：
--
作者：
Fabbrizi MR;Hughes JR;Parsons JL
彗星试验是一种通用、简单、灵敏的凝胶电泳法，可用于测量和准确定量单细胞中的DNA损伤，特别是单链和双链断裂。虽然这通常被用来衡量一组细胞内DNA链断裂水平和修复能力的变化，但这项技术最近已被适应并演变成对特定DNA损伤的更复杂的分析和检测，例如氧化的嘌呤和嘧啶，这是通过利用细胞裂解后的损伤特异性DNA修复酶实现的。在这里，我们详细介绍了一种用于特异性检测复杂DNA损伤(CDD)的酶修饰中性彗星(EMNC)试验，CDD的定义是DNA 1-2螺旋旋转内的两个或更多DNA损伤。CDD诱导与电离辐射(IR)特别相关，特别是与增加线性能量转移(LET)有关，由于IR的修复困难，CDD诱导与IR的细胞杀伤效应有关。因此，EMNC分析揭示了IR诱导的DNA损伤的程度和复杂性的重要细节，但也有可能研究其他可能诱发CDD的遗传毒性因素。
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.