Matti Laaksonen

Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples

A method based on three-DNA-component, sandwich hybridization has been 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 two 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 two 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 shown to be dependent on both 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 X 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.

Nucleic acid sandwich hybridization in adenovirus diagnosis.

The usefulness of viral diagnosis is critically dependent on the rapidity by which it is able to detect and identify virus taken from the site of infection. The methods suitable for rapid diagnosis so far available are mostly based on immunological detection of the virus and its antigenic components (for a review, see Halonen et al., this volume). An alternative to these techniques worth considering is provided by nucleic acid hybridization techniques to detect virus-specific nucleic acid sequences in the sample (for principles and applications see reviews by Bornkamm et al. and Pettersson et al., this volume).

Radio-sequence analysis of in vivo multilabeled nonstructural protein ns86 of Semliki Forest, virus.

Abstract The N-terminal structure of in vivo multilabeled virus-specific enzymes and other proteins can be determined by single radio-sequence analyses. We now show this for Semliki Forest virus nonstructural protein ns86. The viral proteins were labeled with mixtures of up to seven different tritiated amino acids in cultures of chick embryo cells infected with the is-1 mutant. The multilabeled ns86 was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently degraded together with a carrier protein in a liquid phase sequencer. The resulting amino acid derivatives from each cycle of degradation were then separated by high-performance liquid chromatography and the radioactivity of each fraction was determined. The sequence obtained is sufficient to localize the nucleotide sequence on the 42 S RNA genome that codes for the N-terminus of ns86. Considerable differences were found in the incorporation of the labeled amino acids into ns86 during the in vivo conditions used. Stabilities and chromatographic separations of the amino acid derivatives vary and require extra precautions in radio-sequencing of multilabeled proteins.