Improved rapid amplification of cDNA ends (RACE) for mapping both the 5' and 3' terminal sequences of paramyxovirus genomes.
Rapid amplification of cDNA ends (RACE) is a powerful PCR-based technique for determination of RNA terminal sequences. However, most of the RACE methods reported in the literature are developed specifically for the mapping of eukaryotic transcripts with 3' poly-A tail and 5' cap structure. In this study, an improved RACE strategy was developed which allows both 5' and 3' RACE of paramyxovirus genomic RNA using the same set of common molecular biology reagents without having to rely on expensive RACE kits. Mapping of RNA genome terminal sequences is an essential part of characterizing novel paramyxoviruses since these sequences contain important signals for genome replication and transcription, and are important molecular markers for studying virus evolution. The usefulness of this strategy was demonstrated by rapid characterization of both genome ends for a novel paramyxovirus recently isolated from human kidney primary cells. The RACE strategy described in this paper is simple, cost-effective and can be used to map genome ends of any RNA viruses.
Li
Zhuo
Z
Renal Division and Institute of Nephrology, Peking University First Hospital, Beijing, China.
Yu
Meng
M
Zhang
Hong
H
Wang
Hai-Yan
HY
Wang
Lin-Fa
LF
D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D018098:Paramyxovirinae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D020079:Terminal Repeat Sequences / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral