Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence.
Removal of nucleobases from the DNA backbone leads to the formation of abasic sites. The rate of abasic site formation is significantly increased for chemically damaged nucleobases. Thus, abasic sites serve as general biomarkers for the quantification of DNA damage. Herein, we show that capillary electrophoresis with laser-induced fluorescence (CE-LIF) can be used to detect the amount of abasic sites with very high sensitivity. For proof of concept, DNA was incubated with methylmethane sulfonate (MMS) and the damaged bases were removed by incubation at 80 degrees C. The resulting abasic sites were then tagged with a fluorescent aldehyde-reactive probe (FARP). The DNA was precipitated with ethanol, and then analyzed by CE-LIF. CE-LIF and HPLC analysis shows that the fluorescently tagged DNA (DNA-FARP) had a peak area directly proportional to the amount of N-7 methyl guanines. The CE-LIF method had a detection limit of 1.2 abasic sites per 1,000,000 bases or ca. 20 attomoles of abasic sites. This provides a general method for detecting DNA damage that is not only faster but also has comparable or better sensitivity than the alternative ELISA-like method.
Fundador
Erwin
E
Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060, USA.
Rusling
James
J
D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004252:DNA Mutational Analysis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D004247:DNA