RNA · DNA hybridization on membrane filters with fragmented mammalian DNA

Abstract

The possibilities of using fragmented mammalian DNA for hybridization on membrane filters were investigated. The adsorption and release of fragmented DNA were studied as influenced by various factors of the hybridization procedure. It was found that working with preparations sufficiently homogeneous in molecular weight with fragment size 4.8–6.5 S, dissolved in 6 × SSC at neutral pH, the adsorption on filters was almost 100%. After incubation of filters for 18 h in 2 × SSC at 65°C about 50% of the fragmented DNA and 20% of the high molecular weight DNA were released. The degree of release differed for the different families of repeated DNA sequences. Lowest release was obtained with the highly repeated DNA (20%) and highest with the unique DNA (63%), i.e. the release was inversely proportional to the renaturation rate of DNA. In the course of release of fragmented total DNA the material remaining on the filters became enriched in highly repeated sequences, due to selective release of the slowly reassociating fractions. As a result, the percentage of fragmented DNA which hybridized with heterogeneous nuclear RNA was higher than that of high molecular weight DNA. The thermal stabilities of the hybrids with fragmented and high molecular weight DNA were identical. The conditions are defined which permit application of the membrane filter hybridization technique to fragmented mammalian DNA.