Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples.
Long RT-PCR (LRP) amplification of RNA templates is sometimes difficult compared to long PCR of DNA templates. Among RNA templates, hepatitis C virus (HCV) represents an excellent example to challenge the potential of LRP technology due to its extensive secondary structures and its difficulty to be readily cultured in vitro. The only source for viral genome amplification is clinical samples in which HCV is usually present at low titers. We have created a comprehensive optimization protocol that allows robust amplification of a 9.1 kb fragment of HCV, followed by efficient cloning into a novel vector. Detailed analyses indicate the lack of potential LRP-mediated recombination and the preservation of viral diversity. Thus, our LRP protocol could be applied for the amplification of other difficult RNA templates and may facilitate RNA virus research such as linked viral mutations and reverse genetics.
Fan
Xiaofeng
X
Division of Gastroenterology and Hepatology, Saint Louis University Liver Center, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO 63110, USA. fanx@slu.edu
Xu
Yanjuan
Y
Di Bisceglie
Adrian M
AM
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D016679:Genome, Viral / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016415:Sequence Alignment
D018076:DNA, Complementary; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase