Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for in situ hybridization using CARD amplification
Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for in situ hybridization using CARD amplification
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DOI：
10.1177/002215549804600611
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发表时间：
1998-06-01
影响因子：
3.2
通讯作者：
Speel, EJM
中科院分区：
文献类型：
--
作者：
Hopman, AHN;Ramaekers, FCS;Speel, EJM
介绍了一种一步法合成不同酰胺缀合物的方法，该方法可用于催化报告沉积（CARD）扩增系统。生物素、地高辛、荧光素、罗丹明、氨基甲基coumarine乙酸和Cy3的琥珀酰亚胺酯与酪胺在二甲基甲酰胺（DMF）中偶联，用三乙胺（TEA）调节pH为7.0-8.0。偶联反应可在2小时内完成，反应混合物无需进一步的纯化步骤即可应用。此外，在TEA碱化的MilliQ/DMF或NaHCO3 （pH 9.5）缓冲液中，将2,4,6，-三硝基苯磺酸加入酪胺中，制备了三硝基苯(TNP)-酪胺。随后的tnp -酰胺沉淀导致该偶联物的高产分离。所合成的酰胺偶联物已成功应用于单靶点和多靶点原位杂交（ISH）技术，高效检测细胞制剂中的重复和单拷贝DNA靶序列。所描述的方法提供了一种简单和快速的方法，以相对低的成本批量制备各种酰胺缀合物。
A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.0-8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of the TNP-tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single-and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a Variety of tyramide conjugates in bulk amounts at relatively low cost.