ABSTRACT
Transfer of DNA (from 0.1 to 10 micrograms) from diluted solutions of variable volumes (1-10 ml) and various composition (2 M NaCl; 4 M LiCl, 8 M urea; 4 M CsCl; 20% sucrose) to nitrocellulose or nylon membranes was achieved with the use of hydroxyapatite. This absorbent that binds nucleic acids effectively and independently of ionic strength and composition of solution (except for chelators and phosphate ions) easily dissolves in small volumes of acids (for example, in 10% TCA). This phenomenon provides the opportunity to deliver the acid-insoluble precipitates to membrane filters. After alkaline denaturation on the filter followed by a fixation step (baking or UV irradiation for nitrocellulose or nylon filters, respectively), DNA hybridizes effectively with nick-translated DNA probes. The method is simple, reproducible, sensitive, and useful for working with diluted DNA solutions containing interfering substances.
Subject(s)
Collodion , DNA/genetics , Nucleic Acid Hybridization , Nylons , Animals , Chromatography , Cloning, Molecular , DNA/isolation & purification , Durapatite , Genes , Histones/genetics , Hydroxyapatites , Multigene Family , Sea Urchins , SolutionsABSTRACT
Overall DNA-protein interactions in animal cells undergo drastic changes coupled with cellular transitions from quiescence to growth and reversely as revealed by nucleoprotein-Celite chromatography. DNA of chromatin was found to exist in one of the two sharply distinct alternative forms, namely, either tightly or weakly bound to protein moiety. These forms are specific for cycling and quiescent cells, respectively. The tight DNA-protein interactions characterize all cycling cells independent of the cell cycle phase. Transition of DNA of cycling cells from one form to another was observed as a result of treatment of isolated nuclei with DNase I.
