Materials and Reagents

1. Transfection HBV 1.2mer replicons
   
   1. Huh7 cells (Liver Research Center, Rhode Island Hospital, Brown Medical School, Providence, USA)
      
   2. Dulbecco modified Eagle medium (DMEM) (Life Technologies, Gibco^®, catalog number: 11995 )
      
   3. Fetal bovine serum (FBS) (Life Technologies, Gibco^®, catalog number: 26140-079 )
      
   4. Penicillin-streptomycin (Life Technologies, Gibco^®, catalog number: 15140 )
      
   5. Lipofectamine 2000 (Life Technologies, Invitrogen^TM, catalog number: 11668-019 )
      
   6. Opti-MEM^® I Reduced Serum Medium (Opti-MEM) (Life Technologies, Gibco^®, catalog number: 31985-088 )
      
   7. HBV 1.2mer replicon [WT or Drug-resistant HBV, WT HBV1.2 replicon was kindly provided from Prof. W. S. Ryu (Yonsei University, Korea)]
      
   8. Lamivudine (1~100 μM, provided by GlaxoSmithKline)
      
   9. Adefovir (1~100 μM, provided by Gilead Sciences)
      
   10. Clevudine (1~100 μM, provided by Bukwang Pharmaceutical Co.)
       
   11. Entecavir (0.05~1 μM, Moravek)
       
   12. Tenofovir (1~100 μM, provided by Gilead Sciences)
   
   
2. Extraction of viral DNA from HBV core particles
   
   1. DNaseI (Takara Bio Company, catalog number: 2215B )
      
   2. MungBean Nuclease (Takara Bio Company, catalog number: 2420B )
      
   3. Proteinase K (Roche Diagnostics, catalog number: 03115801001 )
      
   4. Phenol-chloroform-isoamyl alcohol 25:24:1 (Sigma-Aldrich, catalog number: 77617 )
      
   5. Sodium acetate trihydrate (NaOAc) (Sigma-Aldrich, catalog number: 236500 )
      
   6. HEPES lysis buffer (see Recipes)
      
   7. Nuclease buffer (I) (see Recipes)
      
   8. 26% PEG solution (see Recipes)
      
   9. Nuclease buffer (II) (see Recipes)
      
   10. 0.5% SDS solution (see Recipes)
   
   
3. Southern blot assay and the construction of the HBV probe
   
   1. Certified Molecular Biology Agarose (Bio-Rad Laboratories, catalog number: 161-3102 )
      
   2. Amersham Hybond-N+ membrane (GE Healthcare, catalog number: RPN203B )
      
   3. SSC Buffer (Sigma-Aldrich, catalog number: 85639 )
      
   4. Salmon sperm DNA (Life Technologies, Invitrogen^TM, catalog number: AM9680 )
      
   5. Poly ethylene glycol (PEG) (Sigma-Aldrich, catalog number: P5413 )
      
   6. 50x Denhardt’s solution (Sigma-Aldrich, catalog number: D2532 )
      
   7. 0.1 M sodium phosphate buffer (pH 7.0)
      
   8. Formamide (Sigma-Aldrich, catalog number: F7503 )
      
   9. HBV 1.2mer replicon
      
   10. Klenow fragment and 10x Klenow buffer (Takara Bio Company, catalog number: 2140B )
       
   11. dNTP (NEB, catalog number: N0446S )
       
   12. [α-³²P] dCTP (3,000 Ci/mmole, Perkin Elmer, catalog number: BLU513H500UC )
       
   13. Quick Spin Columns for radiolabeled DNA purification (Sephadex G-50, Roche Diagnostics, catalog number: 11 273 973 001 )
       
   14. Aat II (New England BioLabs, catalog number: R0117S )
       
   15. Hind III (New England BioLabs, catalog number: R0104S )
       
   16. Random primer (Bioneer, catalog number: N-7052 )
       
   17. Na₂HPO₄ (Amresco, catalog number: 0404 )
       
   18. NaH₂PO₄ (Amresco, catalog number: 0571 )
       
   19. 0.1 M sodium phosphate buffer (see Recipes)
       
   20. Transfer solution (I) (see Recipes)
       
   21. Transfer solution (II) (see Recipes)
       
   22. Transfer solution (III) (see Recipes)
       
   23. Hybridization solution (see Recipes)
       
   24. Washing solution (I) (see Recipes)
       
   25. Washing solution (II) (see Recipes)

Equipment

1. 37 °C, 5% CO₂ forced-air incubator (Thermo Fisher Scientific, Forma^®, model: 3131 )
   
2. 100 mm cell culture dish (Nunc^®, catalog number: 172958 )
   
3. 6 well plate (Nunc^®, catalog number: 140675 )
   
4. Centrifuge (Eppendorf, catalog number: 5415R )
   
5. Pipet Aid XP (Drummond Scientific Company)
   
6. Electrophoresis system (Nihon Eido, catalog number: NB-1011 )
   
7. Dry oven
   
8. Hybridization bottle
   
9. Hybridization chamber
   
10. Heat block
    

Procedure