Quantification of Radiation-Induced Single-Strand Breaks in Plasmid DNA using a TUNEL/ELISA-Based Assay

Abstract

To accurately quantify the number of single-strand breaks (SSBs) induced in plasmid DNA molecules after irradiation, a new type of assay methodology has been explored. The new method is based on the TUNEL (terminal deoxynucleotide transferase dUTP nick end-labeling) assay that was adopted for use under ELISA (enzyme-linked immunosorbent assay) conditions. The assay was found to both improve the quantification and reduce the uncertainties in measurement of SSBs compared with the commonly used agarose gel electrophoresis (AGE) method. Together with AGE, the new method can provide the additional data necessary for an accurate analysis of both SSB and double-strand break (DSB) formation in DNA molecules after irradiation. Furthermore, since only small amounts of DNA are required, the ELISA method can be used to quantify the damage in samples of DNA that are smaller than those required for AGE analysis. As an example of the data obtainable using the new method, plasmid DNA samples were irradiated with vacuum-ultraviolet (VUV) light in an aqueous solution at 170 nm and subsequently analyzed by ELISA. The results were compared directly with those from AGE analysis. The ELISA gave results for SSBs that were an order of magnitude higher than those from AGE and suggested that DSBs are more likely to be the result of two SSBs rather than a single event and that a damaged molecule is more likely to be susceptible to VUV light than an undamaged one.

Low-Dose X-Ray Irradiation Promotes Osteoblast Proliferation, Differentiation and Fracture Healing

“…A variety of types of DNA damage, including single-strand breaks, double-strand breaks and base damage, occur in response to radiation, and the DNA repair process is activated to address such damage. However, high-dose radiation can induce extensive DNA damage that is difficult to repair and might interfere with cell cycle progression, at which point cells undergo programmed cell death [16], [17], [31]. Adaptive protection against DNA damage and bystander effects might also occur in response to low-dose radiation, generally in terms of the protective mechanisms associated with hormesis [11], [15], [32], [33].…”

Radiosensitization of DNA in presence of Pt(II)-based compounds

“…This type of plasmid has already been widely used in irradiation studies [16,17]. NaOH was chosen to stabilize plasmid DNA under vacuum conditions as it was shown that a lack of cations in the vicinity of phosphate backbone in DNA solution prior to solvent evaporation leads to DNA strand breaks and is due to constituent water removal from DNA [18].…”

Evaluating experimental molecular physics studies of radiation damage in DNA*

“…Such a simplification can be used in case of low P S values and it was shown that for investigations of hydroxyl radical attack [55] that this condition was fulfilled, thus the model may be implemented for DNA damage modelling. In addition, when this simplified case is being analysed, and the distribution of SSBs between two DNA strands is not being distinguished but treated as equally distributed between the two stands, the accumulation of DSBs as a result of SSBs accumulation, P DS , can be written as [20] …”

“…Therefore, to accurately determine the level of SSBs in samples that have been exposed to VUV radiation, an alternative analysis method based on the TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labelling) assay, which is used for SSB labelling with a fluorescent probe in cellular DNA to detect apoptotic cells [69] was developed [55]. In this work, the assay has been translated into an ELISA (Enzyme-Linked ImmunoSorbent Assay) -based conditions, in which purified DNA immobilised on plates can be used instead of cells.…”

“…In the first application of the TUNEL/ELISA methodology to molecular physics studies, VUV light-induced damage of DNA (using 170 nm light derived from a synchrotron source) was investigated [55]. By comparing the results obtained from TUNEL/ELISA with results obtained using AGE, it was then possible to explore and confirm the assumption that more SSBs are being produced in the DNA after irradiation than can be assessed with AGE.…”