SANDWICH HYBRIDIZATION AS A CONVENIENT METHOD FOR THE DETECTION OF NUCLEIC-ACIDS IN CRUDE SAMPLES
SANDWICH HYBRIDIZATION AS A CONVENIENT METHOD FOR THE DETECTION OF NUCLEIC-ACIDS IN CRUDE SAMPLES
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DOI：
10.1016/0378-1119(83)90149-x
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发表时间：
1983-01-01
期刊：
影响因子：
3.5
通讯作者：
SODERLUND, H
中科院分区：
文献类型：
--
作者：
RANKI, M;PALVA, A;SODERLUND, H
A method based on 3 DNA-component, sandwich hybridization was designed for the detection and quantitation of nucleic acids in crude samples using adenovirus DNA as a model. Two non-overlapping restriction fragments of adenovirus type 2 (Ad2) DNA were cloned into 2 vectors, the pBR322 plasmid and M13 phage. The recombinant plasmid DNA was immobilized onto nitrocellulose filters and the single-stranded recombinant phage DNA was labeled with 125I and used as a probe. When these 2 reagents were incubated under annealing conditions no radioactivity became filter-bound; only if denatured adenovirus DNA was added as the third reagent, it mediated the attachment of the radioactive probe to the filters. Hybridization efficiency was dependent on the filter and probe DNA concentrations and on the hybridization conditions. When standardized, the assay is quantitative, and under the conditions used 0.2 ng of adenovirus DNA (8 .times. 10-6 pmol) could be detected by an overnight incubation. The test is suitable for crude samples, e.g., solubilized cell extracts, without any purification steps. Less than 100 cells infected with Ad2 can be detected, implying that the assay could be applicable to virus diagnostics.
