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.
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.
Kajino
K
K
Third Department of Internal Medicine, Ehime University School of Medicine, Japan.
Ohta
Y
Y
Miyamura
T
T
Saito
I
I
D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D015698:Genomic Library; D006515:Hepatitis B virus / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D008099:Liver / Q000737:chemistry* / Q000382:microbiology; D009710:Nucleotide Mapping; D012367:RNA, Viral / Q000737:chemistry*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014779:Virus Replication
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012367:RNA, Viral; D004247:DNA; D004256:DNA Polymerase I; D015719:Single-Strand Specific DNA and RNA Endonucleases