Full-length open reading frame amplification of hepatitis C virus

Ulrik Fahnøe; Jens Bukh

doi:10.1007/978-1-4939-8976-8_5

Full-length open reading frame amplification of hepatitis C virus

Ulrik Fahnøe, Jens Bukh^*

^*Corresponding author af dette arbejde

6 Citationer (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.

Originalsprog	Engelsk
Titel	Hepatitis C Virus Protocols
Antal sider	7
Forlag	Humana Press
Publikationsdato	2019
Sider	85-91
ISBN (Trykt)	978-1-4939-8975-1
ISBN (Elektronisk)	978-1-4939-8976-8
DOI	https://doi.org/10.1007/978-1-4939-8976-8_5
Status	Udgivet - 2019

Navn	Methods in Molecular Biology
Vol/bind	1911
ISSN	1064-3745

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10.1007/978-1-4939-8976-8_5

Citationsformater

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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.",

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

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language = "English",

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AU - Fahnøe, Ulrik

AU - Bukh, Jens

PY - 2019

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N2 - 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.

AB - 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.

KW - Cloning

KW - Full-length ORF RT-PCR

KW - HCV

KW - Hepatitis C virus

KW - Long PCR

KW - Next-generation sequencing

KW - RNA extraction

KW - Sanger sequencing

U2 - 10.1007/978-1-4939-8976-8_5

DO - 10.1007/978-1-4939-8976-8_5

M3 - Book chapter

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SN - 978-1-4939-8975-1

T3 - Methods in Molecular Biology

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EP - 91

BT - Hepatitis C Virus Protocols

PB - Humana Press

ER -

Full-length open reading frame amplification of hepatitis C virus

Abstract

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