Northern lights assay: a versatile method for comprehensive detection of DNA damage

Abstract

DNA damage assays have various limitations in types of lesions detected, sensitivity, specificity and samples that can be analyzed. The Northern Lights Assay (NLA) is based on 2D Strandness-Dependent Electrophoresis (2D-SDE), a technique that separates nucleic acids based on length, strandness, structure and conformation changes induced by damage. NLA is run on a microgel platform in 20–25 min. Each specimen is analyzed in pairs of non-digested DNA to detect single- and double-stranded breaks (DSBs) and Mbo I-digested DNA to detect other lesions. We used NLA to evaluate DNA in solution and isolated from human cells treated with various genotoxic agents. NLA detected and distinguished between single- and DSBs, interstrand and intrastrand DNA crosslinks, and denatured single-stranded DNA. NLA was sufficiently sensitive to detect biologically relevant amount of DNA damage. NLA is a versatile, sensitive and simple method for comprehensive and simultaneous analysis of multiple types of damage, both in purified DNA and in DNA isolated from cells and body fluids. NLA can be used to evaluate DNA quality in biosamples, monitor complex molecular procedures, assess genotoxicity, diagnose genome instability, facilitate cancer theranostics and in basic nucleic acids research.

Exploring the SSBreakome: genome‐wide mapping of DNA single‐strand breaks by next‐generation sequencing

“…The traditional comet assay has been adapted to two-dimensional (2D) gel electrophoresis in order to distinguish the signals from SSBs and DSBs (two-tailed comet assay, 2T-Comet [21,22]). When used on bulk purified DNA, separation of DNA by length, strandness, Bulk DNA Single-cell structure and shape via 2D gel electrophoresis is known as the Northern Lights assay [23,24]. Finally, a simplified variation of the comet assay is the halo assay, where damaged DNA is liberated from embedded nuclei by passive diffusion, thus forming a 'halo' whose area is proportional to the amount of damage [25,26].…”

Single-molecule approaches for DNA damage detection and repair: A focus on Repair Assisted Damage Detection (RADD)

“…SSBs and DSBs are commonly measured indirectly by unwinding of the DNA. This includes the comet assay and other electrophoresis-based techniques [9] , [10] , [11] , [12] , [13] . Alternatively, immunological labeling of damage sites using fluorescent-based detection assays such as Enzyme-Linked Immunosorbent Assay (ELISA), Dot-blot, flow cytometry, and immunohistochemistry, have also been utilized [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] .…”

Rapid Quantification of Oxidation and UV Induced DNA Damage by Repair Assisted Damage Detection-(Rapid RADD)

“…Antibodies against specific damage lesions are used for colorimetric or fluorescent-based detection of both single and double strand DNA damage markers. Among the methods utilizing this approach are enzyme-linked immunosorbent assays (ELISA), Dot-blot, flow cytometry, and immunohistochemistry. − The physical integrity of DNA due to single or double strand breaks (SSB and DSB) can be quantified by assays utilizing DNA unwinding such as the comet assay and other electrophoresis based techniques. − Despite the wide acceptance of these methods, DNA damage detection remains challenging mostly due to lack in sensitivity and poor reproducibility. Moreover, in clinically relevant syndromes, minor changes in the amounts of DNA damage lesions can lead to severe outcomes. , Addressing such changes requires high sensitivity and quantitative analysis not easily attained by existing methods.…”