Tilly J L, Hsueh A J
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305-5317.
J Cell Physiol. 1993 Mar;154(3):519-26. doi: 10.1002/jcp.1041540310.
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.
本文描述了一种结合电泳DNA分级分离和放射自显影优势的方法，用于定性和定量分析凋亡（即“程序性细胞死亡”）过程中发生的核小体间DNA片段化。该程序利用末端转移酶将一分子[α-32P] -双脱氧核苷酸均匀地添加到DNA片段的3'末端。经过凝胶电泳和放射自显影分析后，可以对掺入低分子量DNA部分的放射性标记总量进行定量，并用于估计任何给定样品中凋亡DNA片段化的程度。该方法只需15 ng总细胞DNA，与广泛使用的溴化乙锭染色方法相比，将凋亡DNA检测的灵敏度提高了至少100倍。该程序对于分析微量组织和培养细胞中的凋亡应该是有价值的。
