Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques.
The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D004247:DNA / Q000032:analysis; D004277:DNA, Single-Stranded / Q000032:analysis; D003851:Deoxyribonucleases; D004996:Ethidium; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000032:analysis*; D008297:Male; D008854:Microscopy, Electron; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D051381:Rats
D004277:DNA, Single-Stranded; D004247:DNA; D003851:Deoxyribonucleases; D004996:Ethidium