Quantification of radiation-induced single-strand breaks in plasmid DNA using a TUNEL/ELISA-based assay.
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.
Smiałek
Małgorzata A
MA
Department of Physics and Astronomy, The Open University, Walton Hall, Milton Keynes, United Kingdom. smialek@mif.pg.gda.pl
Moore
Sharon A
SA
Mason
Nigel J
NJ
Shuker
David E G
DE
D002138:Calibration; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000528:radiation effects*; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D020287:In Situ Nick-End Labeling / Q000379:methods*; D010957:Plasmids / Q000528:radiation effects*; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded