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 Original Articles |
| Labeling of 3'-Termini of Human ds-RNAs by 5'-32P-pCp Using RNA Ligase |
| Kwang-Sup Lee, Kyung-Hee Kim, Yang Ja Cho |
| Department of Microbiology, Hanyang University, College of Medicine, Seoul, Korea |
Vol.22 Num.3 (p125~129)
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Worldwide, rotavirus (Rv) is the major viral agent of severe diarrhea in children and the current focus of intense research to develop an effective vaccine.
The genome of Rv consists of 11 segments of double-stranded RNA that can be separated by polyacrylamide gel electrophoresis (PAGE). Although detection of Rv in clinical specimens by PAGE has become a routine procedure for a molecular epidemiologic research, it has remained difficult to reveal the migration pattern of 11 Rv segments in the stool specimens with quantities not sufficient.
In this study, some of those specimens with low contents of viral RNA that PAGE with silver staining could not detect (specimens positive by the ELISA but negative by PAGE with silver staining: 24 speciment) were further evaluated by terminal labeling. Purified viral genomic RNA was labeled at the 3'terminus with cytidine 3', 5'-[5'32P] bisphosphate (Amersham, Arlington Heights, III). by using a T4 RNA ligase. The 3'-end-labeled RNA seg,emts were se[arated by PAGE and visualized by means of autoradiography at -70℃.
Only one tenth amount of purified RNA segmets used in a page with silver staining was loaded on a page with a terminal labeling, the page with a terminal labeling demonstrated RNA.
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