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

Maria Rita Fabbrizi,Jonathan R Hughes,Jason L Parsons

doi:10.3390/mps4010014

Maria Rita Fabbrizi, Jonathan R Hughes + Show 1 more

Open Access

https://doi.org/10.3390/mps4010014

Copy DOI

Abstract

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.

Highlights

Over the past decade, the single-cell gel electrophoresis, or comet assay, has become one of the standard methods for assessing DNA damage, with applications in genotoxicity testing, human biomonitoring, and molecular epidemiology, as well as fundamental research in DNA damage and repair, mainly due to its simplicity, sensitivity, versatility, speed and cheapness
complex DNA damage (CDD) is a major factor involved in the cell-killing effects of ionising radiation, as this persists in cells post-treatment and is significantly more difficult to repair than isolated
We demonstrate that the enzyme-modified neutral comet (EMNC) assay can clearly be used to quantify the levels of CDD (DSB-associated) in cultured cells in the absence and presence of irradiation, and could be utilized for the assessment of other DNA damaging agents for their ability to induce CDD

Summary

Introduction

The single-cell gel electrophoresis, or comet assay, has become one of the standard methods for assessing DNA damage, with applications in genotoxicity testing, human biomonitoring, and molecular epidemiology, as well as fundamental research in DNA damage and repair, mainly due to its simplicity, sensitivity, versatility, speed and cheapness. When the negative DNA supercoiling is subsequently unwound by the relatively neutral pH buffer (pH = 9.5), this causes the loops expanded out from the nucleoid core following gel electrophoresis to form a comet tail, visualized using ethidium bromide staining and fluorescence microscopy. The comet assay has proven to be one of the most versatile methods for studying cellular DNA repair capacity, as it allows to quantitatively measure the actual DNA damage induced, as well as the damage remaining at intervals after treatment, allowing a study of the kinetics of cellular repair [5,6]. Since the study of the kinetics of DNA damage repair can be quite laborious and time-consuming using traditional comet assay techniques, we have recently described a variation of this method by treating cells in a suspension of medium with a genotoxin, embedding the cells within an agarose matrix, and allowing the cells to repair the DNA damage in situ in a humidified chamber [7], which will be described

Methods

Results

Conclusion