Chan A, Diamandis E P, Krajden M
Department of Clinical Biochemistry, Toronto Hospital, Ontario, Canada.
Anal Chem. 1993 Jan 15;65(2):158-63. doi: 10.1021/ac00050a012.
We have 5'-end-labeled one polymerase chain reaction (PCR) primer with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). After performing PCR in the presence of another unlabeled primer, we separated the 362 bp PCR product with 2% low melting point agarose gel electrophoresis. The gel was then immersed into a Eu3+ solution. During soaking, Eu3+ diffuses into the gel and associates with BCPDA to form a fluorescent complex of long fluorescence lifetime. This complex can be quantified by scanning the gel with a time-resolved fluorometric reader. Because BCPDA and Eu3+ are not fluorescent by themselves, background signals are very low. The detection limit was about 5 ng of DNA. We have also shown that the BCPDA-labeled product could be blotted and detected on the membrane by using an anti-BCPDA antibody. These two technologies may find applications other than in PCR, e.g. in fluorescence-based DNA sequencing and in solution hybridization.
我们用铕螯合剂4,7-双（氯磺苯基）-1,10-菲咯啉-2,9-二羧酸（BCPDA）对一条聚合酶链反应（PCR）引物的5'端进行了标记。在另一条未标记的引物存在下进行PCR后，我们用2%的低熔点琼脂糖凝胶电泳分离出362 bp的PCR产物。然后将凝胶浸入Eu3+溶液中。浸泡过程中，Eu3+扩散到凝胶中并与BCPDA结合形成具有长荧光寿命的荧光复合物。该复合物可通过时间分辨荧光读数器扫描凝胶进行定量。由于BCPDA和Eu3+本身不发荧光，背景信号非常低。检测限约为5 ng DNA。我们还表明，用抗BCPDA抗体可以在膜上对BCPDA标记的产物进行印迹和检测。这两种技术可能会有除PCR之外的其他应用，例如基于荧光的DNA测序和溶液杂交。
