Latt S A
Proc Natl Acad Sci U S A. 1973 Dec;70(12):3395-9. doi: 10.1073/pnas.70.12.3395.
Fluorescence of the dye 33258 Hoechst bound to chromosomes is partially quenched by the introduction of BrdU into chromosomal DNA. This finding has allowed microfluorometric detection of DNA synthesis in human metaphase chromosomes. Incorporation, shortly before cell harvest, of a pulse of thymidine into chromosomes otherwise substituted with BrdU results in sharply defined foci of bright 33258 Hoechst fluorescence, corresponding to regions of late DNA replication. The latereplicating X chromosome can thereby be clearly identified, and the time course of appearance of its fluorescent bands can be compared with that of its earlier replicating homologue. Growth of cells in medium containing BrdU for two generations allows fluorometric documentation of the semiconservative distribution of newly replicated DNA between sister chromatids, and regions of sister chromated exchange are demarcated. This approach should constitute, in many instances, a convenient, high-resolution fluorometric alternative to autoradiography.
与染色体结合的染料33258 Hoechst的荧光会因溴脱氧尿苷（BrdU）引入染色体DNA而部分淬灭。这一发现使得对人类中期染色体中的DNA合成进行显微荧光测定成为可能。在细胞收获前不久，将胸腺嘧啶脉冲掺入原本用BrdU替代的染色体中，会产生清晰界定的明亮33258 Hoechst荧光焦点，对应于晚期DNA复制区域。由此可以清楚地识别后期复制的X染色体，并将其荧光带出现的时间进程与其早期复制的同源染色体进行比较。在含有BrdU的培养基中培养细胞两代，可以通过荧光测定记录新复制的DNA在姐妹染色单体之间的半保留分布，并划定姐妹染色单体交换区域。在许多情况下，这种方法应该构成一种方便的、高分辨率的荧光测定法，可替代放射自显影术。
