Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.
SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.
D002478:Cells, Cultured; D002482:Cellulose; D004279:DNA, Viral / Q000302:isolation & purification / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D011134:Polysaccharides; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D012367:RNA, Viral / Q000302:isolation & purification*; D012685:Sepharose; D013539:Simian virus 40 / Q000032:analysis*; D014908:Triticum / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis
D004279:DNA, Viral; D011134:Polysaccharides; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D002482:Cellulose; D012685:Sepharose