Generation of digoxigenin-labelled double-stranded and single-stranded probes using the polymerase chain reaction.
As the polymerase chain reaction (PCR) can be used for the generation of vector-free probes, the optimum conditions for incorporation of digoxigenin-11-dUTP into hepatitis B virus (HBV) probes have been investigated. High yields of double-stranded or single-stranded probes can be obtained by utilizing a pair of primers or one primer alone. The probes were tested by dot-blot hybridization on HBV plasmid DNA, slot-blot hybridization on total cellular RNA of Alexander cells and Southern blot hybridization on cellular DNA of Alexander cells and HBV plasmid DNA. They were also tested by in situ hybridization (ISH) on HBV-positive biopsy liver tissue. A ratio of dig-dUTP:dTTP of 1:3 gave highest sensitivity in DNA hybridization. No loss of amplification efficiency and sensitivity was observed when the final concentration of dig-11-dUTP and dTTP was reduced to 20 microM and 60 microM respectively, compared to 200 microM each of dATP, dCTP, dGTP. Several different sizes of double-strand probes were compared by dot-blot hybridization. Longer probes were more sensitive. Strong signal could also be obtained by combination of two or three small probes, which have overlapping sequences. Single-stranded DNA probes had advantages of simplicity of use, high sensitivity and strand specificity.
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Nuffield Department of Pathology and Bacteriology, University of Oxford, John Radcliffe Hospital, UK.
Franklin
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Fleming
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D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D004247:DNA; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral / Q000032:analysis*; D004076:Digoxigenin; D006509:Hepatitis B / Q000382:microbiology*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008099:Liver / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004247:DNA; D004076:Digoxigenin