Breaks in DNA reduce the molecular weight of a single DNA strand, and this may be caused by physical, chemical or enzymatic reagents (6). DNA breaks and lesions may be detected by PCR or using agarose gel electrophoresis (7).

PCR is one of the most frequently used techniques for detecting DNA damage (7). DNA amplification is stopped at the sites of damage via the blocking of the progression of Taq polymerase, which results in a decrease in the quantity of PCR product and a reduced number of DNA templates, which do not contain the Taq-blocked lesions as they are not amplified (8). This is considered to be a simple and reliable method in which particular segments of DNA are specifically replicated and visualized using agarose gels that resolve a range of DNA fragments (50–50,000 bp) dependent on the agarose percentage (8).

Quantitative PCR (qPCR) has been performed to quantify the amount of DNA damage on both strands, as well as the kinetics of DNA damage removal in the mitochondrial DNA (mtDNA) of human and other organisms (7,9). The technique has been used to measure the formation and repair of UV-induced photoproducts in a 1.2-kb fragment of the LacI gene from Escherichia coli (8) and to measure the damage to mtDNA in Schizosaccharomyces pombe cells treated with hydrogen peroxide (10). The frequency of cisplatin-induced lesions has been investigated in a series of fragments ranging from 150 to 2,000 bp from the hamster aprt gene (11). Taken together, these previous studies have demonstrated the ability to detect and analyze gene-specific DNA damage and repair with PCR (12). The qPCR method is dependent on high-molecular weight DNA, DNA quantification, qPCR conditions, quantification of amplification products and the calculation of lesion frequencies (8), and has the advantage of quantitative detection of DNA damage in a specific gene that is expressed mathematically in terms of lesions per kb and the requirement of only 1–2 ng of total genomic DNA (9).

Ligation-mediated PCR (LMPCR) analyzes the distribution of the two types of UV-induced DNA photoproducts, namely cyclobutane pyrimidine dimers and 6–4 photoproducts. The technique has the capability to detect an individual DNA photoproduct at low UV doses (10–20 J/m²) and is also highly sensitive for studying the interactions of proteins and DNA in vivo (13), and for measuring the repair of cyclobutane pyrimidine dimers (14). By contrast, terminal transferase-dependent PCR (TDPCR) is a technique that adds a terminal transferase prior to ligation to an oligonucleotide, and as with LMPCR, this method is able to map pyrimidine 6–4 pyrimidone photoproducts and obtain information on the in vivo chromatin structure (15).

Immuno-coupled PCR (ICPCR) combines nucleic acid amplification with an antibody-based assay in which the detection enzyme in the ELISA is replaced with a biotinylated reporter DNA bound to an antigen-antibody complex (16). This methodology allows for the quantification of thymine dimer formations in genes and these have been established to be directly proportional to the global levels identified in UV radiation-exposed human genomic DNA (17). PCR-based short interspersed DNA element (SINE)-mediated is also a highly sensitive assay that detects DNA adducts produced by drug treatment, including cisplatin (18) or UV-B induced damage, and detects repair in the mammalian genome (19). This assay relies on the abundance, dispersion and conservation of the SINEs in mammalian genomes (19). Compared with conventional PCR and qPCR, this method differs in that it involves the amplification of long segments of DNA in the transcribed regions of the genome in a faster and more cost-effective manner (18).

The comet assay, also known as single-cell gel electrophoresis, is simple and is considered to be one of the gold standard methods for measuring DNA strand breaks (single or double) in eukaryotic cells (34,35). In addition to being a method for detecting DNA breaks, it is also possible to detect UV-induced pyrimidine dimers, oxidized bases and alkylation damage following the introduction of lesion-specific endonucleases (36).

This technique identifies the head of the comet as a spherical mass of undamaged DNA, and the damaged DNA (DNA loops around strand breaks) streams out from the head as a tail (37,38). The comet structure was first described in a study by Ostling and Johanson (39), which explained the tail in terms of DNA with relaxed supercoiling. In the most frequently performed type of comet assay, cells are embedded in agarose to immobilize the DNA and a lysis process is performed using a detergent and high salt. The comet assay has a limited resolution of 10–800 kb using standard conditions (40). Other variants of the comet assay are also used to assess DNA damage and its detection.

This version of the comet assay uses alkaline denaturation surrounding a DNA break to reveal the break (single or double) (41). This method enhances comet tails and extends the range of DNA damage that is detected, but sensitivity has not been increased compared to the use of lesion-specific enzymes (34).

This is a variant of the comet assay that uses an alkaline treatment, after which the conditions are restored to neutral, followed by gel electrophoresis in neutral or mild alkaline conditions (42). This method is less sensitive but remains able to detect SSBs (43).

The use of lesion-specific enzymes may aid in the detection of other types of DNA damage, other than SSBs or DSBs, including oxidized bases or pyrimidine dimers (44). The enzymes create an apurinic/apyrimidic site by removing the damaged base; endonucleases specifically detect oxidized pyrimidines, and formamidopyrimidine DNA glycosylases detect 8-oxo-7,8-dihydroguanine and ring opened-purines (35).

This technique combines a comet assay and FISH, and is effective in detecting damage and repair site-specific breaks in DNA regions in individual cells (40). This assay may be used to measure and discriminate between SSBs or DSBs or modifications from DNA repair.

This technique is based on the intercalation of PI into the DNA helix, which causes the DNA to become a supercoiled structure (45). Following lysis, the nucleoids of individual cells appear as ‘halos’ that correspond to DNA loops, which may be measured to determine the chromatin fragility. The ‘halo’ diameter is proportional with PI concentration and is expressed as relaxed or rewound supercoils at low PI and high PI, respectively (45). This method may aid the study of the effects of induced DNA damage, although it only detects alterations in the organization of DNA if the damage has not been repaired, which occurs at radiation doses of 2 Gy. This assay has limitations on its sensitivity, but the advantages are that it is able to measure the DNA damage of a single cell and no labeling of DNA with radioactive precursors is required (46).

The TUNEL assay detects SSBs or DSBs, as well as levels of apoptosis via the visualization of DNA fragmentation (45). This assay primarily uses the ability of the enzyme TdT to incorporate nucleotide analogues conjugated with a fluorochrome onto the free 3′-OH of a DNA strand, therefore allowing the visualization of the nuclei that contain fragmented DNA (47). Additionally, fluorescence may be detected using a fluorescent dye conjugated antibody that recognizes biotin- or digoxigenin-tagged nucleotides (48). As the assay is able to detect the DNA fragments with fluorescence or radioactivity, microscopy techniques, FCM, photo-multipliers and charge coupled device arrays may be used to detect and quantify DNA damage caused by apoptosis (49). Typically, the visualization of DNA damage is possible as the morphological alterations occur in the nucleus, including alterations in structural organization and the collapse of chromatin (49). During the degradation of DNA, a specific pattern of fragments is generated by the activity of endonucleases enzymes, and fragmentation of genomic DNA occurs into lower molecular weight fragments from DNA (47).

Although this method was designed for detecting DNA damage following apoptosis, DNA fragments with 3′-OH ends may occur in a number of other situations where apoptosis does not take place, including necrosis (49). The TUNEL assay is limited in its sensitivity and specificity, but it may also be used to stain cells undergoing DNA repair (50). TUNEL is not considered sufficient to establish the type of cell death and must be accompanied by another method that allows for the distinction of the origin of the DNA fragmentation in cells undergoing apoptosis or non-apoptotic DNA damage (51). One of the assays that is considered to specifically detect DNA DSBs and used in combination with TUNEL assay is the in situ ligation assay (52), which is based on ligation of double-stranded oligonucleotide probes by T4 DNA ligase to the ends of the DNA breaks directly in tissue sections (53).

FISH is a technique for the visualization of nucleic acids that improves resolution, speed and safety compared with older methods that use isotopic detection (54,55). This technology also allowed for the development of simultaneous detection of multiple targets, quantitative analyses and live-cell imaging (54). FISH is typically used to locate and examine chromosomal, genetic and genomic aberrations that are associated with the development and progression of disease (56). Therefore, it has clinically important applications in cytogenetic and oncology, including in identifying gene alterations in patients with cancer (56). A modification of this technique, DBD-FISH, has been used to investigate cervical cancer progression by detecting and quantifying DNA breaks in genomic regions that are sensitive to destabilization (57). This technique allows detection and quantification of SSBs and DSBs in the genome or in a specific DNA sequence from a single cell (58). There are certain disadvantages in fluorescence assays, including the reproducibility and irregularity of the signals, and background autofluorescence (54).

When DNA breakage occurs, it is important to differentiate between necrosis, autolysis and apoptosis (59). FCM was developed to detect apoptosis (60); this method allows for the measure of a large number of cells, and is also used to detect DNA strand fragmentation, chromosomal aberrations and chemical adducts in DNA (61,62).

Annexin V protein is used to quantify the number of dead or apoptotic cells (63). The lipid bilayer in healthy cells does not allow for Annexin V binding, however, in cells undergoing apoptosis, Annexin V binds to the outer surface of the cell membrane following translocation of phosphatidylserine in the presence of Ca²⁺ (64). The number of apoptotic cells may be quantified using FCM (65). With the use of a secondary antibody tagged with FITC or PI, this method may detect important proteins involved in DNA repair complexes (27). FCM is able to rapidly and sensitively measure DNA damage compared with the frequently used comet assay method.

The RIA binding assay is used to measure the concentration of antigens using specific antibodies. The target antigen is synthesized with a radiolabel and without a label, and is subsequently bound to specific antibodies (66). Following the introduction of a sample, a competitive reaction develops between the radiolabeled antigens and the unlabeled antigens from the sample, and this releases an amount of radiolabeled antigen. Standard curves may be obtained from this process by mixing equal amounts of antibody and radiolabeled antigen, with increasing concentrations of non-labeled antigen in a constant volume; unknown antigen is similarly mixed with antibody and radiolabeled antigen, and the concentration may be subsequently determined (67). This assay may be used to estimate the quantity of 6–4 photoproducts and cyclobutane dimers in DNA (45).

This is one of the most commonly used immunological methods for the quantification of DNA damage (67) and consists of affixing an unknown quantity of antigen to a surface and applying an unknown quantity of antibody to the surface so that the antibody binds to the antigen. The antibody is linked to an enzyme that may be quantified via the addition of an appropriate substrate (colored, fluorescent or radioactive) (45,67).

This assay utilizes fixed cells that have previously been treated with proteases and RNase. This process removes proteins and RNA, and this ensures that cross-reaction with DNA does not occur (67). A solution of PI is used to counterstain the cells. The resulting immunofluorescence allows for visualization of the nuclei in adduct-negative cells (45). Immunohistochemical assays, in addition to FISH, have served as a more effective screening and diagnostic tool to detect alterations in certain metabolites, including the case of ALK gene in non-small cell lung cancer (68).

This technique measures the presence of oxidative DNA via the immunoslot-blot system, and uses chemiluminescent detection and secondary antibodies that are conjugated to alkaline phosphatase enzymes and radioactive iodine (69). This assay is effective, but is limited by the cross-reactivity of the antibodies with normal DNA bases.

Oxidative stress and absorption of UV light by nucleic acids has been established to be one of the causes of oxidative DNA damage, which may promote cancer development (70,71). The improvement of HPLC coupled to tandem MS with an electrospray ionization mode, may be a sensitive and accurate method to detect modified bases of the oxidative-damaged DNA and UV-induced dimeric pyrimidine photoproducts (72). Notably, during the initial steps of the BER, the simultaneous detection and quantification of altered and released nucleobases from genomic DNA may be conducted using HPLC-MS (73). Therefore, this technique may be useful for detecting SSBs, as these lesions and base alterations are involved with proteins of the BER pathway (74).

This assay has been used to quantify oxidized nucleosides, including 8-oxo-7,8-dihydro-2′-deoxyguanosine, 8-oxo-7,8-dihydro-2′-deoxyadenosine, 5-formyl-2′-deoxyuridine, 5-hydroxymethyl-2′-deoxyuridine, 5-hydroxy-2′-deoxyuridine and the four diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine within isolated and cellular DNA following exposure to γ-rays (75). It is also possible to detect tandem DNA lesions as dinucleoside monophosphates, and in addition to detecting the type of DNA damage, HPLC-MS may also provide information on the location and quantity of DNA damage (75,76). Despite the advantage of accuracy, this assay has the limitations of a high cost and the large amount of experience that is required to accurately use the technique to monitor the formation of low levels of oxidized bases within cellular DNA (75). However, it remains the method of choice for measuring modified DNA bases.

To understand diverse cellular processes, including DNA damage, repair and its biological consequences, it is important to characterize and quantify DNA lesions.

MS provides structural evidence for a biological or chemical analysis, and in combination with gas chromatography, it enables measurements of more complex samples (77). GC-MS is a technique capable of measuring numerous products of DNA damage, including those of the sugar moiety and heterocyclic bases, as in HPLC-MS (78). The MS analysis provides sensitive detection of a single DNA lesion in DNA with multiple lesions or nucleobases following chemical or enzyme degradation of the nucleic acids (79). Additionally, this technique measures the kinetics of a number of DNA repair enzymes and is able to identify and quantify the expression levels of DNA repair proteins in human tissues (80,81). Typically, these measurements include the hydrolysis of DNA, the derivatization of hydrolysates and the separation via gas chromatography of hydrolysates that are identified and quantified using MS (78). GC-MS has also been used to identify DNA-protein crosslinks, including Thy-Gly, Thy-Ala and Cyt-Tyr, in mammalian chromatin in vitro (82–84).

It has been established that DNA may be damaged by reactive oxygen species and the alterations in DNA that are formed are detected using electrochemical methods based on the inherent sensitivity of DNA-mediated charge transport (CT). These methods are also capable of detecting base pair mismatches and the majority of base damage products (85). This methodology may detect DNA-mediated CT as a damage detection mechanism for DNA repair enzymes (86). There have been hypotheses regarding the development of a sensor for the detection of single base mutations and DNA base lesions in duplex DNA to utilize the sensitivity of this charge to transport DNA films (87). The electrochemical method, electrocatalysis, has provided the basis for novel assays to detect low levels of lesions and possible for use as an early diagnostic tool. Although this is a method that provides sensitive, selective and low cost detection of DNA damage, it has the limitation of not being able to recognize thymidine dimer lesions until they are connected with the distortion of DNA double helix (45).