A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry.
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 safety transported prior to analysis.
Gong
J
J
Cancer Research Institute, New York Medical College, Valhalla 10595.
Traganos
F
F
Darzynkiewicz
Z
Z
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001853:Bone Marrow / Q000737:chemistry; D002166:Camptothecin / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000302:isolation & purification* / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D000431:Ethanol; D005434:Flow Cytometry; D006801:Humans; D007938:Leukemia / Q000097:blood / Q000188:drug therapy / Q000473:pathology*; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000473:pathology; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004273:DNA, Neoplasm; D000431:Ethanol; D002166:Camptothecin