Selectivity of RNA chain initiation in vitro. 1. Analysis of RNA initiations by two-dimensional thin-layer chromatography of 5'-triphosphate-labeled oligonucleotides.
A method for the rapid and quantitative analysis of 5'-terminal oligonucleotides of RNAs made in vitro is described. The method involves synthesis of RNA in the presence of [gamma-32P]ATP or GTP, isolation of the RNA, and digestion with T1 or pancreatic ribonucleases to release labeled 5'-triphosphate termanated oligonucleotides. The oligonucleotides are then subjected to chromatography on a polyethyleniminecellulose thin-layer system using 2 M LiCl, 0.01 M EDTA (pH 6.5) in the first dimension and 1.5 M LiCl, 1.8 M formic acid, 0.005 M EDTA (pH 2.0) in the second. RNAs made with E. coli RNA polymerase and lambdacb2, T7, T4, and adenovirus 2 DNA yield characteristic fingerprint patterns. The utility of this method in studying selectivity of in vitro RNA chain initiation is discussed.
D002855:Chromatography, Thin Layer; D003090:Coliphages / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009843:Oligoribonucleotides / Q000032:analysis; D012329:RNA, Bacterial / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D009843:Oligoribonucleotides; D012329:RNA, Bacterial; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases