A vector with transcriptional terminators increases efficiency of cloning of an RNA virus by reverse transcription long polymerase chain reaction.
Full-length cDNA clones of RNA viruses are advantageous for maintaining the genomic sequence without the generation of diversity by accumulation of sequence mutations during productive virus replication. They permit in vitro manipulation of the genomic clone to test the effect of sequence changes on the phenotype of reactivated virus. Infectious cDNA clones have been produced by ligation of subgenomic clones but are sometimes difficult to generate in a single cloning operation. We used reverse-transcription to synthesize full-length cDNA from genomic RNA of Coxsackievirus B3 of the Picornavirus family and enzymatically amplified this by long PCR. Five different cloning vectors were used to clone the long PCR product, including the vector Lorist6 which contains transcriptional terminators on either side of the cloning site to prevent transcription of inserts in E. coli. No recombinant colonies were obtained from any of the vectors lacking transcriptional terminators but three full-length clones were obtained using Lorist6. The results suggest that transcriptional terminators increase the recovery of cDNA clones of the 7.4 kb Coxsackie virus genome in this cosmid vector, without resort to phage packaging, representing an advance over previous methods and advantages in the molecular manipulation of these viruses.
Cameron-Wilson
C L
CL
Cell and Molecular Biology Section, Division of Biomedical Sciences, Imperial College School of Medicine, London, UK.
Zhang
H
H
Zhang
F
F
Buluwela
L
L
Muir
P
P
Archard
L C
LC
D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D029822:Enterovirus B, Human / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013728:Terminator Regions, Genetic; D014709:Vero Cells
D018076:DNA, Complementary