Journal article
Biotinylated and radioactive DNA probes for detection of varicella-zoster virus genome in infected human cells
Molecular and cellular probes, Vol.2(3), pp.197-207
1988
DOI: 10.1016/0890-8508(88)90004-7
PMID: 2851737
Abstract
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 μCi [
32P] and 0·4 μg
Hind III fragment A of the varicella genome, while the second probe employed a biotin-7-dATP analogue and 1·0 μg 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 μg 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 μg total DNA from infected cells. Similar tests on DNA extracted from uninfected cell samples were negative with both probes.
Details
- Title: Subtitle
- Biotinylated and radioactive DNA probes for detection of varicella-zoster virus genome in infected human cells
- Creators
- Gilbert CuthbertsonCharles Grose
- Resource Type
- Journal article
- Publication Details
- Molecular and cellular probes, Vol.2(3), pp.197-207
- Publisher
- Elsevier Ltd
- DOI
- 10.1016/0890-8508(88)90004-7
- PMID
- 2851737
- ISSN
- 0890-8508
- eISSN
- 1096-1194
- Language
- English
- Date published
- 1988
- Academic Unit
- Stead Family Department of Pediatrics; Infectious Disease (Pediatrics)
- Record Identifier
- 9984093472202771
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