Quantitation of DNA Breaks and Application of the Assay.

To quantitate DNA breaks, a method based on saturation labeling 3′ ends of DNA fragments with α 32 P dCTP in the presence of 2′, 3′ dideoxy cytidine 5′ triphosphate (ddCTP) by terminal deoxynucleotidyl transferase (TdT) was developed . The saturation labeling of 3′ ends of DNA fragments was performed by adding different concentrations of α 32 P dCTP to a DNA sample, from which a maximal labeling ( L max ) and a kinetic parameter( K m) of the TdT reaction were calculated. Results were confirmed by agarose gel electrophoresis, fluorescein dUTP and exogenous teminal deoxynucleotidyl transferase(TUNEL), flow cytometric analysis (FCA). The method mentioned above requires as little as 5 ng of DNA, increases in the sensitivity of DNA fragments detection by at least 200 fold relative to the widely used agarose gel electrophoresis, and the linearity of the assay is about 5～5 000 ng DNA. The application of the method in the apoptosis study showed that(1) a time and dose dependent increase in the number of DNA strand breaks in apoptotic Raji lymphoma lymphocytes induced by dexamethasone, and (2) age dependent increase in the number of DNA strand breaks occurred in the cardiac tissues of spontaneously hypertensive rats (SHR) compared with that of normal control rats (WKY). Results of the assay were confirmed by the DNA ladder pattern exhibited after electrophoresis, fluorescein dUTP and exogenous terminal deoxynucleotidyl transferase(TUNEL), flow cytometric analysis(FCA)( r 0 98). It is a quantitative, simple, sensitive, specific and useful assay for assessing DNA degradation in molecular and cell biology especially in apoptosis research.