Amplification and cloning of a long RNA virus genome using immunocapture-long RT-PCR

  1. Romero, Alicia 1
  2. Blanco-Urgoiti, Begoña 2
  3. Ponz, Fernando 1
  1. 1 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria
    info

    Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria

    Madrid, España

    ROR https://ror.org/011q66e29

  2. 2 CIMA-Arkaute, Vitoria, Alava, Spain
Revista:
Journal of Virological Methods

ISSN: 0166-0934

Año de publicación: 1997

Volumen: 66

Número: 1

Páginas: 159-163

Tipo: Artículo

DOI: 10.1016/S0166-0934(97)00043-8 GOOGLE SCHOLAR lock_openAcceso abierto editor

Otras publicaciones en: Journal of Virological Methods

Resumen

A rapid and easy method was developed in order to amplify long fragments of the genome of potato virus Y (PVY), a virus possesing 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.

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