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Full-Length Open Reading Frame Amplification of Hepatitis C Virus

  • Ulrik Fahnøe
  • Jens Bukh
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)

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.

Key words

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious DiseasesCopenhagen University HospitalHvidovreDenmark
  2. 2.Department of Immunology and Microbiology, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

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