Theoretical approaches for detection of sequence-specific Sister-Chromatid Exchanges in interphase cells

Abstract

Two procedures for detection of Sister-Chromatid Exchanges (SCEs) in interphase cells within specific DNA sequence areas are delineated. Proliferating cells cultured with 5-bromodeoxyuridine (BrdU) for one cell cycle are subjected to cytokinesis-block to produce binucleated cells. Then, the BrdU-substituted DNA strands are removed as in the chromosome orientation-FISH (CO-FISH) procedure. In one case, the intact unsubstituted DNA strands may be hybridized with differentially labelled direct and reverse single-stranded DNA (ssDNA) probes from the target area. After formation of a SCE during the first cycle, specifically within the target area, three signals should be visualized in each nucleus of the binucleated cell. Two of them, of different color, must appear colocalized or adjacent simultaneously in both sister nuclei, corresponding to the SCE site, being accompanied by a signal from the other homologous locus showing a complementary color pattern in both sister nuclei. Other possibility is hybridizing two separated direct ssDNA probes flanking the target sequence area labelled with the same color and the two reverse complementary ssDNA probes labelled with other color. The presence of three signals with similar color and one of different color in each nucleus from the binucleated cell, following a complementary color pattern between both nuclei, would indicate the presence of a SCE within the DNA sequence area flanked by the DNA probes. The availability of complementary ssDNA probes specific for single locus should make the Interphase SCE-FISH a suitable procedure.