By Karl Maramorosch, Hilary Koprowski
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Extra resources for Methods in Virology, Volume 8
Cells in throat washings were hybridized to 3H-labeled and biotinylated BamHl V. Figure 5 shows the cytohybridization to epithelial cells from an EBV-seronegative donor (Fig. 5a and b) and to cells from patients with infectious mononucleosis (Fig. 5c-h). The morphological deterioration of the cells in Fig. 5g and h suggests viral cytolysis. 22 N. R A A B - T R A U B A N D J. S. PAGANO FIG. 5. EBV DNA in oropharyngeal epithelial cells by cytohybridization. (a,b) Epithelial cells from the throat washings of an EBV-seronegative donor do not hybridize to either probe, (c-h) The clear nuclear location of hybridization in the epithelial cells of patients with infectious mononucleosis suggests an intracellular location of the virus.
F. OBUESKI have a high statistical probability of containing unique sequences that occur only once in a haploid viral genome. For the hypothetical 8000-base viral genome described in the example above, oligonucleotides containing 10 or more base residues constitute approximately 25% of the total sequences. Although the structurally unique oligonucleotides of a typical RNA mole cule are too numerous to be resolved effectively by a single mechanism of separation, the two-dimensional fingerprinting technique, in which a dif ferent separation mechanism is employed for each dimension, allows good resolution to be achieved in practice.
In Situ HYBRIDIZATION One great advantage of in situ hybridization is the ability to localize virusspecific sequences to a particular cell type. It is also possible to detect viral nucleic acid in a rare cell when biochemical extraction of the nucleic acid from the total sample would have diluted viral sequences beyond detection. The availability of cloned fragments for providing virus-specific probes of reduced complexity and the development of new ways of labeling DNA have greatly increased the sensitivity of these techniques since the work re ported in Vol.