Third-strand in situ hybridization (TISH) to non-denatured metaphase spreads and interphase nuclei
Third-strand in situ hybridization (TISH) to non-denatured metaphase spreads and interphase nuclei
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DOI：
10.1007/s004120050367
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发表时间：
1999-07-01
期刊：
影响因子：
1.6
通讯作者：
Fresco, JR
中科院分区：
文献类型：
--
作者：
Johnson, MD;Fresco, JR
已经开发了一种方法，用于在固定但不另外变性的中期扩散和间期核中，在发现在溶液中最佳的条件下，将寡脱氧核糖核苷酸“第三链”与双链体DNA靶结合。第三链原位杂交（TISH）在pH6.0的pH 17 Z1基因座的人染色体17 α-卫星（D17 Z1）中的一个独特的多拷贝靶序列的pH 17 Z1和生物素修饰的16-核苷酸同嘧啶第三链。UVA-光固定的第三链，呈现荧光异硫氰酸荧光素标记的抗生物素蛋白，可重复的着丝粒特异性染色体17，和可见的淋巴细胞和体细胞杂交传播和间期核的信号没有放大。鉴定了两个第三链特异性D17 Z1单倍型。TISH对天然中期和间期染色质具有潜在的诊断、生物化学和流式细胞术适用性。
A methodology has been developed for binding oligodeoxyribonucleotide 'third strands' to duplex DNA targets in fixed but not additionally denatured metaphase spreads and interphase nuclei under conditions found to be optimal in solution. Third-strand in situ hybridization (TISH) at pH 6.0 of a psoralen- and biotin-modified 16-nucleotide homopyrimidine third strand to a unique 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-specific for chromosome 17, and visible without amplification of the signal in lymphocyte and somatic cell hybrid spreads and interphase nuclei. Two third-strand-specific D17Z1 haplotypes were identified. TISH has potential diagnostic, biochemical, and flow cytometric applicability to native metaphase and interphase chromatin.