Wilhelm J, Pingoud A, Hahn M
Justus-Liebig-Universität Giessen, Germany.
Biotechniques. 2001 May;30(5):1052-6, 1058, 1060 passim. doi: 10.2144/01305rr04.
In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in LightCycler assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.
在定量实时聚合酶链反应（PCR）分析中，使用荧光团标记的寡核苷酸探针来产生用于定量评估的序列特异性信号。虽然TaqMan探针在PCR过程中必须被Taq DNA聚合酶的内切核酸酶活性水解以产生信号，但LightCycler分析中的杂交探针不能被水解。在本研究中，我们针对四个不同的靶标证明了探针在PCR过程中会被Taq DNA聚合酶降解。使用缺乏内切核酸酶活性的Stoffel片段和抑制非特异性产物形成的TaqStart抗体，可以提高信号产量、扩增曲线质量和定量测量的准确性，而无需费力优化扩增方案。
