Fractionation of DNA from mammalian cells by alkaline elution

Abstract

The method of alkaline elution provides a sensitive measure of DNA single-strand length distribution in mamalian cells and is applicable to a variety of problems concerning DNA damage, repair, and replication. The physical basis of the elution process was studied. The kinetics of elution above the alkaline transition pH were found to occur in two phases: an initial phase in which single-strand length is rate limiting, followed by a phase in which elution is accelerated due to the accumulation of alkali-induced strand breaks. The range of DNA single-strand lengths that can be discriminated by elution above the alkaline transition pH was estimated by calibration relative to the effects of x ray, and was found to be 5 X 10(8)-10(10) daltons. Shorter DNA strands elute within the pH transition zone, which extended from pH 11.3 to 11.7 when tetrapropylammonium hydroxide was used as base. This elution was relatively rapid, but was sharply limited by pH, according to the length of the strands: the length of the strands eluted increased with increasing pH. Alkaline elution was inhibited by treatment of cells with low concentrations of nitrogen mustard, a bifunctional alkylating known to cross-link DNA. On investigation of the possibility that DNA subclasses may differ in their elution behavior, satellite L strands were found to elute more slowly from cells exposed to a low dose of x ray than did the bulk DNA.