Abstract
This chapter describes the procedures for the generation of recombinant hepatitis B virus (HBV) that contains the polymerase/reverse transcriptase (pol/RT) gene from clinical isolates. It also contains procedures for phenotypic testing of the recombinant HBV in HepG2 cells for susceptibility to nucleos(t)ide analogs. Amplification and cloning of the pol/RT gene as opposed to the full-length HBV genome from a clinical isolate allows for clinical samples with lower viral loads to be successfully analyzed. Furthermore, it enables a direct comparison of sequence changes in the pol/RT region between isolates.
MeSH terms
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Cell Culture Techniques
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Cloning, Molecular
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DNA Replication
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DNA, Viral / genetics
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DNA, Viral / isolation & purification
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Genes, pol*
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Genetic Vectors / genetics
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Genetic Vectors / isolation & purification
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Hep G2 Cells
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Hepatitis B virus / isolation & purification
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Hepatitis B virus / physiology*
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Humans
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Multiplex Polymerase Chain Reaction
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Phenotype*
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Plasmids / genetics
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Plasmids / isolation & purification
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Polymerase Chain Reaction
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RNA-Directed DNA Polymerase / genetics*
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Real-Time Polymerase Chain Reaction
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Recombination, Genetic*
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Virus Replication
Substances
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DNA, Viral
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RNA-Directed DNA Polymerase