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In the present study, we
describe a method of quantifying DNA fragmentation. This assay is based
on saturation labeling 3`-ends of DNA fragments with [alpha]32PdCTP in the
presence of 2`,3`-dideoxy-cytidine-5`-triphosphate (ddCTP) by terminal deoxynucleotidyl
transferase (TdT). The saturation labeling of 3`-ends of DNA fragments was
performed by adding different concentrations of [alpha]32PdCTP to a DNA
sample, from which a maximal labeling (Lmax) and a kinetic parameter (Km)
of the TdT reaction are calculated. The saturated labeling gives true quantitation
that makes it possible to accurately compare quantities of DNA fragments
among different samples. This method requires as little as 5 ng of DNA and
increases the sensitivity of apoptotic DNA detection by at least 200-fold
relative to the widely used ethidium staining method. The application of
this method in an apoptosis study showed that (a) a time- and dose-dependent
increase in the number of DNA strand breaks in apoptotic lymphocytes was
induced by dexamethasone, and (b) age-dependent apoptosis occurred in the
cardiac tissues of spontaneously hypertensive rats. Results of this assay
were confirmed by the DNA ladder pattern exhibited after electrophoresis
as well as the morphologic changes of apoptosis observed under electron
microscopy and were very consistent with results obtained in quantifying
apoptotic cells by flow cytometric analysis (r = 0.98, p = 0.002). Thus,
this assay is quantitative, simple, sensitive, and useful for assessing
apoptosis. |
