Prentice D A, Gurley L R
Biochim Biophys Acta. 1983 Jun 24;740(2):134-44. doi: 10.1016/0167-4781(83)90070-2.
Experiments using nucleases as probes of chromatin structure take place in two stages: (1) nuclei isolation, and (2) nuclease digestion. The parameters of the nuclease digestion stage are usually strictly controlled because of nuclease sensitivity to them. However, there have been no reports on whether parameters in the nuclei isolation stage affect the subsequent nuclease digestions. We have evaluated a typical nuclei isolation technique with respect to how changes in the isolation parameters affect nuclease digestion kinetics. Our observations point out that various parameters encountered in the nuclei isolation stage have a significant effect on the subsequent nuclease digestion kinetics of DNAase I. These parameters include the concentration of cells, divalent cations and phosphatase inhibitors. The pH, concentration of NaCl and concentration of detergent had little effect. Micrococcal nuclease was relatively unaffected by changes in the nuclei isolation parameters. The importance of this report lies in the demonstration that lack of control of seemingly insignificant parameters, such as cell concentration during the nuclei isolation stage, leads to subsequent irreproducible results in the DNAase I digestion. These findings indicate that great care must be exercised in the nuclei isolation stage if reproducible work is to be performed with DNAase I.
（1）细胞核分离，以及（2）核酸酶消化。由于核酸酶对其敏感，核酸酶消化阶段的参数通常受到严格控制。然而，关于细胞核分离阶段的参数是否会影响后续核酸酶消化，尚无相关报道。我们评估了一种典型的细胞核分离技术，研究分离参数的变化如何影响核酸酶消化动力学。我们的观察结果指出，细胞核分离阶段遇到的各种参数对随后的DNA酶I核酸酶消化动力学有显著影响。这些参数包括细胞浓度、二价阳离子和磷酸酶抑制剂。pH值、NaCl浓度和去污剂浓度影响较小。微球菌核酸酶相对不受细胞核分离参数变化的影响。本报告的重要性在于证明，对看似微不足道的参数缺乏控制，如细胞核分离阶段的细胞浓度，会导致随后DNA酶I消化结果不可重复。这些发现表明，如果要用DNA酶I进行可重复的工作，在细胞核分离阶段必须格外小心。
