Efficient cloning of hepatitis B virus DNA from single-stranded replicative intermediates and its application to S1 mapping of viral RNA in human liver tissue
✍ Scribed by Kazunori Kajino; Yasuyuki Ohta; Tatsuo Miyamura; Izumu Saito
- Publisher
- John Wiley and Sons
- Year
- 1991
- Tongue
- English
- Weight
- 836 KB
- Volume
- 33
- Category
- Article
- ISSN
- 0146-6615
No coin nor oath required. For personal study only.
✦ Synopsis
An efficient method for cloning subgenomic fragments of the hepatitis B virus (HBV) was developed, utilizing its abundant single-stranded replicative intermediates. The total genomic DNA obtained from the liver tissue of patients with chronic HBV infection was treated by using the Klenow fragment of E. coli DNA polymerase I without adding any exogenous primers. Single-stranded replicative intermediates were efficiently converted to double-stranded linear DNA, one end of which terminated at (or near to) the direct repeat 1 (DR1) sequence of the HBV genome. By screening less than 1,000 recombinants from a DNA library after this treatment, we obtained a subgenomic HBV fragment of 2.0 kilobases. We then analysed HBV RNA in human liver tissue by S1 mapping. It was possible to map HBV RNA only when a DNA probe from the same tissue was used.