During the past few years, several methods have been developed for the detection of specific nucleic acid sequences by in situ hybridization using non-radioactive labels such as fluorochromes, cytochemically detectable enzymes and electron-dense markers. These methods are preferable to autoradiography in terms of speed of performance and topological resolution. Their limited sensitivity, however, has so far restricted their use to the detection of repeated sequences. Here we report single gene detection with a procedure using 2-acetylaminofluorene (AAF)-modified probes, immunoperoxidase cytochemistry and reflection-contrast microscopy. We confirmed the autoradiographic data on the localization of the human thyroglobulin (Tg) gene to the distal end of the long arm of chromosome 8. A mixture of cosmid cHT2-derived subclones of the 3' part of the Tg gene, 22.3 kilobase pairs (kbp) in total, was used as a hybridization probe. This procedure can be used to map other unique sequences, if genomic clones are available from which clones with an appropriate amount of inserts can be isolated.
A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the procedure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of non-radioactive in situ hybridizations.
SUMMARY
To obtain objective criteria for the diagnosis of incipient mycosis fungoides, Feulgen‐DNA cytophotometry was carried out in skin imprint preparations from patients with clinically definite mycosis fungoides and from patients suspected of having this disease. Cell imprints of benign skin lesions such as chronic dermatitis served as controls.
All patients with mycosis fungoides showed abnormal histograms with aneuploid and polyploid Feulgen‐DNA distribution patterns. In the control group the mean class of Feulgen‐DNA values was diploid.
Five per cent or more of non‐diploid cells was taken as a criterion for malignancy. On this basis, eight of the fifteen patients in the suspect group showed abnormal histograms. Six of these eight patients developed a malignant skm reticulosis within a year. The other (seven) patients in this group showed normal histograms, and none of them developed a skin malignancy during a follow‐up period of 2½ years.
Feulgen‐DNA cytophotometry may therefore be considered an objective aid in the diagnosis of the early stages of mycosis fungoides.
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