Third-strand in situ hybridization (TISH) to non-denatured metaphase spreads and interphase nuclei

A methodology has been developed for binding oligodeoxyribonucleotide 'thir d strands' to duplex DNA targets in fixed but not additionally denatured me taphase spreads and interphase nuclei under conditions found to be optimal in solution. Third-strand in situ hybridization (TISH) at pH 6.0 of a psora len- and biotin-modified 16-nucleotide homopyrimidine third strand to a uni que multicopy target sequence in human chromosome 17 alpha-satellite (D17Z1 locus) is described. UVA-photofixed third strands, rendered fluorescent by fluorescein isothiocyanate-labeled avidin, are reproducibly centromere-spe cific for chromosome 17, and visible without amplification of the signal in lymphocyte and somatic cell hybrid spreads and interphase nuclei. Two thir d-strand-specific D17Z1 haplotypes were identified. TISH has potential diag nostic, biochemical, and flow cytometric applicability to native metaphase and interphase chromatin.