Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX, 78712, USA.
Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX, 78712, USA.
Anal Biochem. 2021 Sep 15;629:114239. doi: 10.1016/j.ab.2021.114239. Epub 2021 May 9.
Analysis of catalytic activity of nucleic acid enzymes is crucial for many applications, ranging from biotechnology to the search for antiviral drugs. Commonly used analytical methods for quantifying DNA and RNA reaction products based on slab-gel electrophoresis are limited in throughput, speed, and accuracy. Here we report the optimization of high throughput methods to separate and quantify short nucleic acid reaction products using DNA sequencing instruments based on capillary electrophoresis with fluorescence detection. These methods afford single base resolution without requiring extensive sample preparation. Additionally, we show that the utility of our system extends to quantifying RNA products. The efficiency and reliability of modern instruments offers a large increase in throughput but complications due to variations in migration times between capillaries required us to develop a computer program to normalize the data and quantify the products for automated kinetic analysis. The methods presented here greatly increase sample throughput and accuracy and should be applicable to many nucleic acid enzymes.
分析核酸酶的催化活性对于许多应用至关重要，从生物技术到抗病毒药物的研究都有涉及。目前常用的基于板凝胶电泳的分析方法在通量、速度和准确性方面存在局限性。在这里，我们报告了使用基于毛细管电泳和荧光检测的 DNA 测序仪来分离和定量短核酸反应产物的高通量方法的优化。这些方法提供了单碱基分辨率，而不需要进行广泛的样品制备。此外，我们还表明，我们的系统可用于定量 RNA 产物。现代仪器的效率和可靠性可大大提高通量，但由于需要在毛细管之间进行迁移时间的变化，这使得操作变得复杂，因此我们开发了一个计算机程序来对数据进行标准化，并对产物进行定量，以进行自动动力学分析。这里提出的方法大大提高了样品的通量和准确性，并且应该适用于许多核酸酶。
