An improved method for the isolation of the inverted repetitive (foldback) sequences present in mammalian DNAs is described. It makes use of the new observation that nuctease Sl digestion of denatured DNA occurred at a faster rate and was more extensive in medium containing dioxaue. The temperature-absorbance characteristics of nuclease Sl-resistant DNA were systematically studied as a function of the temperature employed during the step of enzymic hydrolysis. Specimens of human pIacentaf and calf thymus DNA which had been denatured and renatured to cd < lOma mol s Iiter-' were used as substrates. Fotdback DNA was isolated from the enzymic digests by means of hydroxylapatite chromatogtaphy. Temperature-absorbance studies showed the enzyme-resistant DNA had a high degree of thermal stability; the hyperchromic rises equated those obtained in the native specimens. The amount of foldback DNA which could be obtained was not influenced by the fragment size of the starting material, above a certain moiecuhtr weight range. Fofdback DNA represented about 4% of the human genome and at least 5% of the bovine genome. The size distributions of these strands were studied by means of polyacrylamide gel etectrophoresis.
' Abbreviations used: HA, Hydroxylapatite; AZD, acetate-zinc-dioxane medium, prepared by mixing 1 vol of 0.1 M sodium acetate-O.0001 M zinc acetate (pH 5.0) with 0.5 vol of dioxane: SSC, 0.15 M NaCI-0.015 M sodium citrate, pH 7.4; T,,,, the temperature at which the midpoint of the hyperchromic rise is attained; Cd, DNA concentration CM) multiplied by the time (s) allowed for reassociation. tRNA, transfer RNA: L, length.