High throughput quantification of short nucleic acid samples by capillary electrophoresis with automated data processing

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. [Display omitted] •Improved separation of short FAM labeled oligonucleotides by CE.•Customizable Cy3 internal standard for more accurate sizing of short nucleic acids.•New open source software presented to automate data analysis and visualization.•Validation experiments show method is compatible with both DNA and RNA.