UNEXPECTED ELECTROPHORETIC MIGRATION OF RNA WITH DIFFERENT 3' TERMINICAUSES A RNA SIZING AMBIGUITY THAT CAN BE RESOLVED USING NUCLEASE P1-GENERATED SEQUENCING LADDERS

Citation
J. Cruzreyes et al., UNEXPECTED ELECTROPHORETIC MIGRATION OF RNA WITH DIFFERENT 3' TERMINICAUSES A RNA SIZING AMBIGUITY THAT CAN BE RESOLVED USING NUCLEASE P1-GENERATED SEQUENCING LADDERS, Biochemistry, 37(17), 1998, pp. 6059-6064
Citations number
28
Categorie Soggetti
Biology
Journal title
Volume
37
Issue
17
Year of publication
1998
Pages
6059 - 6064
Database
ISI
SICI code
Abstract
It has been widely believed that the electrophoretic migration differe nce of otherwise identical RNAs with a P versus OK terminus would be t he same as occurs for DNA, a fairly reproducible similar to 1/2 nucleo tide (nt) offset. RNA with a 5'-OH indeed migrates less than or equal to 1 nt slower than if it had a 5'-P. Surprisingly, however, RNA with a 3'-OH terminus (generated by many cellular RNases of interest) migra tes anywhere from similar to 1/4 to similar to 2 nts slower than the o therwise identical molecule with a 3'-P or 2',3'-cyclic-P terminus las present on standard RNase-generated sequencing ladders). This previou sly unrecognized variability in electrophoretic migration offset cause s a 1-2 nt ambiguity in a commonly used method of RNA size determinati on. We also show two ways to overcome this problem and enable rigorous sizing of 3'-OH terminating RNAs. Most convenient is to use sequencin g standards generated by nuclease P1, which is generally sequence-nons pecific but we show becomes G-specific or A-preferential under certain reaction conditions.