Lauc G, Perovic S, Dapper J, Flögel M, Iskric S, Müller W E
Department of Medical Biochemistry, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia.
Anal Cell Pathol. 1994 Aug;7(2):107-14.
A new method is described which is suitable for reliably analysing apoptotic fragmentation in small amounts of DNA. After isolation, DNA was labelled with biotin-4-dUTP using Klenow polymerase. Then DNA was size-separated by agarose gel electrophoresis, blot transferred and subsequently visualized by the streptavidin alkaline phosphatase-BCIP/NBT procedure. This non-radioactive method was used to detect apoptotic DNA in rat pheochromocytoma PC12 cells, treated with tributyltin (1 nM). While only 30 ng of DNA is required for analysis of apoptotic DNA using the new blot technique, 100-fold more material is needed to identify the fragmentation of DNA after separation by agarose gel electrophoresis and direct staining with ethidium bromide. In a further set of experiments, rat cortical cells were incubated with human immunodeficiency virus type 1 viral glycoprotein of M(r) of 120 kDa (gp120) to induce apoptosis. More than 0.3 ng of gp 120/ml are required to detect apoptotic DNA by the direct procedure; only 0.1 ng gp120/ml or less were sufficient to document clear DNA fragmentation using the non-radioactive blotting technique described here. These results demonstrate that the new procedure can be used to analyse very small amounts of apoptotic DNA and shows that gp120-induced apoptosis can be measured at low concentrations of the viral protein.
本文描述了一种新方法，该方法适用于可靠地分析少量DNA中的凋亡片段化。DNA分离后，使用Klenow聚合酶用生物素-4-dUTP进行标记。然后通过琼脂糖凝胶电泳对DNA进行大小分离，印迹转移，随后通过链霉亲和素碱性磷酸酶-BCIP/NBT程序进行可视化。这种非放射性方法用于检测用三丁基锡（1 nM）处理的大鼠嗜铬细胞瘤PC12细胞中的凋亡DNA。使用新的印迹技术分析凋亡DNA仅需要30 ng DNA，而通过琼脂糖凝胶电泳分离并用溴化乙锭直接染色来鉴定DNA片段化则需要多100倍的材料。在另一组实验中，将大鼠皮质细胞与人免疫缺陷病毒1型分子量为120 kDa的病毒糖蛋白（gp120）孵育以诱导凋亡。通过直接方法检测凋亡DNA需要超过0.3 ng gp120/ml；使用本文所述的非放射性印迹技术，仅0.1 ng gp120/ml或更低浓度就足以证明明显的DNA片段化。这些结果表明，新方法可用于分析极少量的凋亡DNA，并表明在低浓度病毒蛋白下可测量gp120诱导的凋亡。
