Yu H, Chao J, Patek D, Mujumdar R, Mujumdar S, Waggoner A S
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.
Nucleic Acids Res. 1994 Aug 11;22(15):3226-32. doi: 10.1093/nar/22.15.3226.
Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps.
荧光原位杂交（FISH）已成为研究和临床诊断各个领域不可或缺的工具。许多应用需要一种快速、简单且灵敏的同时检测多个靶序列的方法。在这项工作中，我们描述了两种新的花青染料标记的dUTP类似物Cy3-dUTP和Cy5-dUTP的合成。它们是DNA聚合酶的有效底物，可通过标准缺口平移、随机引物法和聚合酶链反应掺入DNA探针中。已确定最佳标记条件，可产生每千碱基含20 - 40个染料的探针。用这些类似物获得的直接标记DNA探针为多色多序列分析提供了一种简单方法，无需二次检测试剂和步骤。
