Schweighoffer T, Schweighoffer E, Apati A, Antoni F, Molnar G, Lapis K, Banfalvi G
Institute of Biochemistry Department I, Semmelweis Medical University, Budapest, Hungary.
Histochemistry. 1991;96(1):93-7. doi: 10.1007/BF00266767.
DNA staining methods based on aspecific interactions with dye molecules have been replaced by an immunofluorescent approach to measure DNA replication. Biotin-11-dUTP was incorporated into permeable thymocytes isolated after emetine or cyclosporin A treatment of mice. Active sites of DNA replication were amplified based on biotin-avidin interaction and verified under fluorescent microscope. Cytometry of fluorescent images allow the direct measurement of replicating DNA without aspecific detection of total cellular DNA. Cytometric analysis of replication revealed that emetine acts at the early S phase, while cyclosporin A blocks in vivo DNA synthesis at mid S phase.
基于与染料分子非特异性相互作用的DNA染色方法已被一种免疫荧光方法所取代，用于测量DNA复制。在对小鼠进行依米丁或环孢素A处理后，将生物素-11-dUTP掺入分离出的可渗透胸腺细胞中。基于生物素-抗生物素蛋白相互作用扩增DNA复制的活性位点，并在荧光显微镜下进行验证。荧光图像的细胞计数法可直接测量复制中的DNA，而无需对总细胞DNA进行非特异性检测。复制的细胞计数分析表明，依米丁作用于S期早期，而环孢素A在体内阻断S期中段的DNA合成。
