Journal: PLoS Pathogens
Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
Figure Lengend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
Article Snippet: NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs).
Techniques: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing