Amplification and cloning of a long RNA virus genome using immunocapture-long RT-PCR.
A rapid and easy method was developed in order to amplify long fragments of the genome of potato virus Y (PVY), a virus possessing a 10-kb genomic RNA. The method of immunocapture-RT-PCR was adapted, by using thermostable DNA polymerases with proofreading activity and the proper buffers and cycles, to amplify almost the whole genome of PVY in two fragments (5.6 and 4.3 kb) without purifying virions nor viral RNA. Both fragments were cloned subsequently and their ends sequenced. The method is applicable to the rapid cloning and molecular characterization of the genomes of many other RNA viruses.
Romero
A
A
Laboratorio de Biología Molecular y Virología Vegetal, CIT-INIA, Madrid, Spain.
Blanco-Urgoiti
B
B
Ponz
F
F
D003001:Cloning, Molecular; D016679:Genome, Viral; D016133:Polymerase Chain Reaction / Q000379:methods*; D017800:Potyvirus / Q000235:genetics*; D014158:Transcription, Genetic