Abstract
Studying the biogenesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and developing anti-HBV agents require analytical methods to quantify viral DNA levels inside host cells. The well-accepted Southern blotting method is only semi-quantitative, while the other widely used methods (based on qPCR) have been questioned as to their fidelity for cccDNA quantification. In addition, Southern blotting and qPCR results barely reflect the number of host cells present in an analytical sample. We here developed new techniques that substantially improve cccDNA detection and quantification, including a sample pretreatment method that i) exploits high temperature and exonuclease V (an ATP-dependent, bidirectional exonuclease) digestion to effectively increase the amplification efficiency of HBV cccDNA by removing rcDNA and denaturing the cccDNA template, and ii) a method that splits cell samples and uses separate extraction technologies to facilitate “host normalization” based on host genomic DNA signals. Our study introduces new analytical techniques that should be useful for the basic biology and translational studies of HBV.
Published Version
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