Recent developments in methods for RNA sequencing using in vitro 32P-labeling.
A variety of approaches that utilize in vitro 32P-labeling of RNA and of oligonucleotides in the sequence analysis of RNAs are described. These include 1) methods for 5'- and 3'- end labeling of RNAs; 2) end labeling and sequencing of oligonucleotides present in complete T1 RNase or pancreatic RNase digests of RNA; 3) use of random endonucleases, such as nuclease P1, for terminal sequence analysis of end labeled RNAs; and 4) use of base specific enzymes or chemical reagents in the sequence analysis of end-labeled RNAs. Also described is an approach to RNA sequencing, applied so far to tRNAs, which is based on partial and random alkaline cleavage of an RNA to generate a series of overlapping oligonucleotide fragments, all containing the original 3'-end of the RNA. Analysis of the 5'- end group of each of these oligonucleotides (following 5'-end labeling with 32P) provides the sequence of most of the tRNA. The above methods have been used to derive the sequences of several tRNAs, the ribosomal 5S and 5 x 8S RNAs, a viroid RNA, and large segments of both prokaryotic and eukaryotic ribosomal and messenger RNAs.
D001345:Autoradiography; D001483:Base Sequence; D008970:Molecular Weight; D009843:Oligoribonucleotides / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases; D010761:Phosphorus Radioisotopes; D012313:RNA; D011865:Radioisotope Dilution Technique; D012260:Ribonucleases; D013379:Substrate Specificity
D009843:Oligoribonucleotides; D010761:Phosphorus Radioisotopes; D012313:RNA; D012260:Ribonucleases; D010744:Phosphoric Monoester Hydrolases