Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system

Abstract

An aqueous polymer phase system containing 6.3% (wt/wt) dextran and 3.5% (wt/wt) poly(ethylene glycol) in 10 nM phosphate buffer (pH 8.0) was developed to select RNA-DNA hybrids from unhybridized RNA. The top phase of this phase system, which contains DNA and the RNA-DNA hybrids, can be used to purify adenovirus mRNA early and late in the infectious cycle. The hybrids can be melted by heat in the top phase and the mRNA selected by oligo(dT)cellulose chromatography whereupon the polymers and the DNA percolate and the polyadenylated mRNA absorb to the column. The isolated mRNA appears to be almost quantitatively recovered at a purity from 70-90% depending on the concentration of the specific mRNA in the starting material. Early and late viral mRNA were selected on the complementary strands of adenovirus DNA according to this procedure.