Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation.
A method combining the advantages of electrophoretic DNA fractionation and autoradiography is described for the qualitative and quantitative analysis of internucleosomal DNA fragmentation that occurs during apoptosis, or "programmed cell death." This procedure utilizes terminal transferase enzyme to uniformly add one molecule of [alpha 32P]-dideoxynucleotide to the 3'-end of DNA fragments. Following gel electrophoresis and autoradiographic analysis, the total amount of radiolabel incorporated into the low molecular weight DNA fraction can be quantitated and used to estimate the degree of apoptotic DNA fragmentation in any given sample. This method requires as little as 15 ng of total cellular DNA and increases the sensitivity of apoptotic DNA detection by at least 100-fold over the widely used ethidium bromide staining method. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells.
Tilly
J L
JL
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305-5317.
Hsueh
A J
AJ
D000818:Animals; D017209:Apoptosis; D001345:Autoradiography; D001717:Birds; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006107:Granulosa Cells / Q000166:cytology; D007553:Isotope Labeling; D007700:Kinetics; D009707:Nucleosomes / Q000378:metabolism; D006080:Ovarian Follicle / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley
D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D009707:Nucleosomes; C037773:2',3'-dideoxyadenosine triphosphate; D004247:DNA