Full-length open reading frame amplification of hepatitis C virus

Ulrik Fahnøe, Jens Bukh*

*Corresponding author for this work
    6 Citations (Scopus)

    Abstract

    The purpose of this method is to amplify the full coding sequence of hepatitis C virus (HCV) by a single round reverse transcriptase-polymerase chain reaction (RT-PCR) approach. Our method relies on a highly robust and sensitive RNA extraction procedure and cutting-edge RT-PCR enzymes, all of which have been rigorously tested and optimized. This will not only allow for robust amplification of the entire open reading frame (ORF) of HCV for sequencing by Sanger or next-generation sequencing (NGS), but can also be used for cloning of the ORF of uncharacterized samples and for linkage analysis of mutations on individual genomes spanning the entire ORF. The method has been validated on a variety of samples, including sera from HCV patients and cell-culture supernatants.

    Original languageEnglish
    Title of host publicationHepatitis C Virus Protocols
    Number of pages7
    PublisherHumana Press
    Publication date2019
    Pages85-91
    ISBN (Print)978-1-4939-8975-1
    ISBN (Electronic)978-1-4939-8976-8
    DOIs
    Publication statusPublished - 2019
    SeriesMethods in Molecular Biology
    Volume1911
    ISSN1064-3745

    Keywords

    • Cloning
    • Full-length ORF RT-PCR
    • HCV
    • Hepatitis C virus
    • Long PCR
    • Next-generation sequencing
    • RNA extraction
    • Sanger sequencing

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