Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA; Biochemistry and Molecular Biology Track, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA.
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA; Medical Scientist Training Program, Mayo Clinic Graduate School of Biomedical Sciences and Mayo Clinic Alix School of Medicine, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA.
Anal Biochem. 2022 Aug 1;650:114712. doi: 10.1016/j.ab.2022.114712. Epub 2022 May 10.
The quantitative polymerase chain reaction (qPCR) with detection of duplex DNA yield by intercalator fluorescence is a common and essential technique in nucleic acid analysis. We encountered unexpected results when applying standard qPCR methods to the quantitation of random DNA libraries flanked by regions of fixed sequence, a configuration essential for in vitro selection experiments. Here we describe the results of experiments revealing why conventional qPCR methods can fail to allow automated analysis in such cases, and simple solutions to this problem. In particular we show that renaturation of PCR products containing random regions is incomplete in late PCR cycles when extension fails due to reagent depletion. Intercalator fluorescence can then be lost at standard interrogation temperatures. We show that qPCR analysis of random DNA libraries can be achieved simply by adjusting the step at which intercalator fluorescence is monitored so that the yield of annealed constant regions is detected rather than the yield of full duplex DNA products.
荧光染料嵌合法的实时定量聚合酶链反应（qPCR）是核酸分析中常用且必不可少的技术。当我们将标准 qPCR 方法应用于侧翼为固定序列的随机 DNA 文库定量时，遇到了意想不到的结果，因为这种配置对于体外选择实验是必不可少的。在这里，我们描述了实验结果，揭示了为什么在这种情况下，常规 qPCR 方法不能允许自动分析，以及解决此问题的简单方法。特别是，我们表明，当由于试剂耗尽而导致延伸失败时，含有随机区域的 PCR 产物的复性在后期 PCR 循环中不完全，并且在标准检测温度下，荧光染料可能会丢失。我们表明，通过简单地调整监测嵌合染料荧光的步骤，可以实现对随机 DNA 文库的 qPCR 分析，以便检测退火的恒定区域的产量，而不是检测完整双链 DNA 产物的产量。
