Biotinylated and radioactive DNA probes for detection of varicella-zoster virus genome in infected human cells.
We have developed and compared two DNA dot hybridization methodologies with similar probes (radioisotope-labelled and biotin-labelled) to detect varicella-zoster virus (VZV) DNA in three different cell cultures at varying times post-infection. Control cultures included uninfected monolayers of the same cells. Cellular DNA was isolated by a standard phenol extraction method, after which the DNA was quantified, serially diluted and blotted onto nitrocellulose or nylon membranes. The VZV DNA probe, which consisted of the large Hind III A fragment (27 of the total 125 kbp), was produced in two separate nick translation systems. The first contained 100 microCi [32P] and 0.4 microgram Hind III fragment A of the varicella genome, while the second probe employed a biotin-7-dATP analogue and 1.0 microgram of the Hind III fragment A. Direct visualization on the membrane or the exposed radiographic film showed a dot of varying intensity whenever viral genome was detected with either the biotin or the radioactive probe, respectively. With the [32P]-labelled probe, we detected VZV genomic sequences within 0.5 microgram total DNA at 12 h post-infection. This amount corresponded to approximately 5-10 pg of viral DNA. By comparison, hybridization with the biotin-labelled probe required 0.5-1.0 micrograms total DNA from infected cells. Similar tests on DNA extracted from uninfected cell samples were negative with both probes.
Cuthbertson
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G
Department of Pediatrics, University of Iowa College of Medicine, Iowa City 52242.
Grose
C
C
D001710:Biotin; D002460:Cell Line; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003315:Cornea; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005814:Genes, Viral; D014645:Herpesvirus 3, Human / Q000235:genetics*; D006801:Humans; D008545:Melanoma; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis
D015342:DNA Probes; D004279:DNA, Viral; D010761:Phosphorus Radioisotopes; D001710:Biotin