Purification of RNA-DNA hybrids by exclusion chromatography.
A simple method for selection of RNA-DNA hybrids has been developed and applied to the purification of adenovirus-specific messenger RNA. Cytoplasmic RNA prepared from adenovirus type 2 (ad2)-infected HeLa cells or from an ad2-transformed rat cell line was hybridized in solution to the complementary strands of ad2 DNA. The hybridization mixture was subsequently fractionated by chromatography on a Sepharose 2B column. The intact probe DNA as well as the RNA-DNA hybrids are excluded from the gel matrix and elute with the void volume. Nonhybridized RNA, in contrast, is included into the gel matrix and elutes as a broad peak well separated from the excluded fractions. Fractions corresponding to the void volume, were collected and the RNA-DNA hybrids were denatured in 90% formamide. The selected RNA was separated from the DNA by affinity chromatography on poly(U)-Sepharose. Restriction endonuclease fragments of DNA with a large enough size to make them excluded from the agarose column were also used for hybridization. In these experiments hybridizations were carried out under conditions which would allow R-loop formation (Thomas, M., White, R.L., and Davis, R.W. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2294-2298) and the hybridized RNA was separated from unhybridized RNA by Sepharose chromatography. The validity of the method was demonstrated by programming an in vitro protein-synthesizing system with selected RNA.
D000260:Adenoviruses, Human / Q000032:analysis* / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods; D004262:DNA Restriction Enzymes; D004279:DNA, Viral; D006367:HeLa Cells / Q000032:analysis; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism
D004279:DNA, Viral; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes