A new method for quantitative estimation of the degree of DNA fragmentation utilizing agarose gel electrophoresis.
We designed a new method for quantitative analysis of the degree of DNA fragmentation, a characteristic feature of apoptosis. A photograph of an agarose gel electrophoresis of fragmented DNA was incorporated by an optical density scanner or equivalent equipment, and the integrations of middle molecular size area (10,000 to 300 bp), single nucleosomal size area (smaller than 300 bp) and total lane area were calculated. We defined fragmentation rate or (%)FR as the amount of fragmented DNA expressed as the percentage of the total amount of DNA, assigning the coefficients of 1 and 0.5, respectively, to the fragments completely digested into single nucleosomal length and to those partially digested (between 10 k to 300 bp). A standard calibration curve was constructed from triplicate experiments of target nuclei digestion by micrococcal nuclease, which revealed that this method covered a wide range of nuclease activity. We also confirmed that our method was applicable to an autodigestion assay of isolated nuclei which represented endogenous endonuclease activities. This method may be a useful tool for quantitative analysis of endonuclease activities capable of producing nucleosomal-size DNA fragmentation.
Kawabata
H
H
1st Division of Internal Medicine, Faculty of Medicine, Kyoto University, Japan.
Anzai
N
N
Yoshida
Y
Y
Okuma
M
M
D017209:Apoptosis; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D006801:Humans
D004247:DNA