Nuclease digestibility of chromatin is affected by nuclei isolation procedures.
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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002453:Cell Cycle; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000032:analysis; D002843:Chromatin / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology
D002843:Chromatin; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D008274:Magnesium; D002118:Calcium