A SELECTIVE PROCEDURE FOR DNA EXTRACTION FROM APOPTOTIC CELLS APPLICABLE FOR GEL-ELECTROPHORESIS AND FLOW-CYTOMETRY
A SELECTIVE PROCEDURE FOR DNA EXTRACTION FROM APOPTOTIC CELLS APPLICABLE FOR GEL-ELECTROPHORESIS AND FLOW-CYTOMETRY
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DOI：
10.1006/abio.1994.1184
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发表时间：
1994-05-01
影响因子：
2.9
通讯作者：
DARZYNKIEWICZ, Z
中科院分区：
文献类型：
--
作者：
GONG, JP;TRAGANOS, F;DARZYNKIEWICZ, Z
在发生细胞凋亡（程序性细胞死亡）的细胞中，一部分核DNA被分裂成与单核小体或寡核小体中的DNA大小相当。当琼脂糖凝胶电泳分析这种DNA时，它会产生不连续DNA片段的特征“阶梯”模式。这种DNA降解模式通常作为细胞死亡的凋亡模式的标志。我们开发了一种简单、快速、选择性的方法，用于从凋亡细胞中提取降解的低分子量DNA。将细胞用70%的乙醇预先固定，用0.2 M的磷酸柠檬酸缓冲液在pH 7.8下提取DNA，提取液依次用RNase A和蛋白酶K处理，然后进行电泳。从1-2 × 10(6)个HL-60细胞中提取的DNA以及接受化疗的白血病患者的血液和骨髓样本中检测到阶梯模式，流式细胞术标准显示，其中只有8%的细胞凋亡。该方法快速，使用无毒试剂（无酚、氯仿等）。这种方法允许从流式细胞术测量的同一细胞群中提取DNA，以估计DNA倍性、非凋亡细胞的细胞周期分布、凋亡细胞的百分比或其他参数。此外，在分析之前，细胞可以在70%乙醇中储存至少几周，而不会有任何明显的DNA降解。用乙醇处理还可以使几种病原体失活，从而提高样品处理的安全性。该方法适用于临床样品，可在乙醇中固定，然后在分析前储存和/或安全运输。(C) 1994学术出版社，Inc.
In cells undergoing apoptosis (programmed cell death), a fraction of nuclear DNA is fragmented to the size equivalent of DNA in mono- or oligonucleosomes. When such DNA is analyzed by agarose gel electrophoresis it generates the characteristic ''ladder'' pattern of discontinuous DNA fragments. Such a pattern of DNA degradation generally serves as a marker of the apoptotic mode of cell death. We developed a simple, rapid, and selective procedure for extraction of the degraded, low-molecular-weight DNA from apoptotic cells. The cells are prefixed in 70% ethanol, DNA is extracted with 0.2 M phosphate-citrate buffer at pH 7.8, and the extract is sequentially treated with RNase A and proteinase K and then subjected to electrophoresis. The ladder pattern was detected from DNA extracted from 1-2 X 10(6) HL-60 cells, of which as few as 8% were apoptotic, by flow cytometric criteria, as well as from blood and bone marrow samples from leukemic patients undergoing chemotherapy. The method is rapid and uses nontoxic reagents (no phenol, chloroform, etc.). This approach permits the analysis of DNA extracted from the very same cell population that is subjected to measurements by flow cytometry to estimate DNA ploidy, the cell cycle distribution of nonapoptotic cells, the percentage of apoptotic cells, or other parameters. Furthermore, the cells may be stored in 70% ethanol for at least several weeks before analysis without any significant DNA degradation. Treatment with ethanol also inactivates several pathogens, thereby increasing the safety of sample handling. The method is applicable to clinical samples, which can be fixed in ethanol and then stored and/or safely transported prior to analysis. (C) 1994 Academic Press, Inc.