Hydroxyapatite chromatography of short double-helical DNA

Abstract

Short double-helical segments of T7 DNA, ranging in length from 16 to more than 2000 nucleotide pairs, were specifically cleaved by the restriction endonuclease from Hemophilus aegyptius, endonuclease Z. These segments were separated by gel electrophoresis, and assigned lengths on the basis of their mobility using the known lengths of Hin II + III and H. aegyptius endonuclease III segments of ФX replicative form I as a standard. Shorter segments elute from hydroxyapatite at lower concentrations of sodium or potassium phosphate buffer: the concentration of phosphate buffer causing elution is inversely proportional to the reciprocal of the segment length. On the basis of these plots one may estimate that double-helical segments having more than 17 nucleotide pairs will be retained on hydroxyapatite at 25 °C in 0.14 M potassium buffer. When this expectation is tested by prolonged eluting with 0.14 M potassium phosphate buffer, the minimum segment length that is quantitatively retained was found to be between 28 and 37 nucleotide pairs long. Similar experiments with sodium phosphate buffer at 60 °C indicate segments longer than 43 to 54 will be quantitatively retained on hydroxyapatite in spite of prolonged washing with 0.12 M sodium phosphate buffer. When duplex segments are treated with exonuclease III to produce partly single-chained molecules, it is found that the elution position is that expected for single-chain-free duplex segments of the calculated shorter length. This suggests that the presence of single chains has no effect on binding of duplexes to hydroxyapatite.