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

Abstract

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.

Keywords: Capillary electrophoresis; DNA and RNA analysis; DNA polymerase; High throughput; Quantification and analysis software; Sanger sequencer.

Figure 1:. Exonuclease validation experiment for PAGE versus CE.

Scheme: The DNA substrate is a 27 nt, 5′-[6-FAM]-labeled primer annealed to the 45 nt template, with a buried T:T mismatch at the n-2 position. Bases to be removed are shown in red. Reaction conditions: A solution of 1.25 μM T7 DNA polymerase, 25 μM thioredoxin, 0.1 mg/ml BSA, and 12.5 mM Mg²⁺ was mixed with 250 nM FAM-labeled DNA from the other syringe in the quench flow at 20°C to start the reaction. Time points were quenched by mixing with EDTA from the quench syringe to a final concentration of 0.3 M. A) Analysis of time points by denaturing PAGE. Samples were mixed with formamide loading dye, separated by denaturing PAGE with 15% acrylamide/7M urea, and scanned on a Typhoon 9500 scanner with the FAM fluorescence filter. B) Separation of 5 ms time point by CE with POP-7 polymer. The same samples from (A) were injected at 1.6 kV for 15 seconds. Three peaks are visible, however lack of separation between products make quantification difficult. C) Separation of 5 ms time point by CE with POP-6 polymer. The same samples from (A) were injected with the same parameters as in (B). Each product is well resolved to near baseline, facilitating accurate peak integration. D) Overlay of data analyzed by PAGE versus CE. Data for products of varying lengths (27 nt, red; 26 nt, green; 25 nt, blue; 24 nt, yellow) for analysis by PAGE (open circles, dashed line) versus capillary electrophoresis (closed circles, solid line). Net rates and amplitudes obtained by fitting the data to a double exponential burst equation are identical for the two data sets within 2%.

Figure 2:. Single nucleotide incorporation experiment and internal standard normalization

Scheme: The DNA substrate is a 27 nt, 5′-[6-FAM]-labeled primer containing a phosphorothioate linkage at the 3′ end (denoted by *), annealed to a 45 nt template strand. The added nucleotide is shown in red. Reaction conditions: A solution of 75 nM T7 DNA polymerase (exo⁻), 1.5 μM Thioredoxin, 0.1 mg/ml BSA, and 150 nM FAM-DNA was mixed with 100 μM dATP and 12.5 mM Mg²⁺ to start the reaction in the quench flow instrument at 20°C. Time points were quenched by mixing with EDTA from the quench syringe (0.3 M final concentration). A) Electropherogram showing the 15 ms time point with the Cy3 internal standard. Samples were injected for 15 seconds at 2.6 kV. Blue peaks correspond to FAM-labeled reaction products and the yellow peak corresponds to the Cy3-labeled internal standard. X_n is the migration time of the peak for DNA n nucleotides in length. X_IS is the migration time of the Cy3 labeled internal standard oligo. The formula for the corrected migration time for the 27 nt peak is shown in the panel and is used to normalize the variable migration times between capillaries. B) Plot of DNA concentration versus time for varying length products derived from the burst experiment. The 27 nt primer is shown in red and the 28 nt primer is shown in green as a function of time. This representative data set is part of the included example data provided with the software. Both sets of data are shown fit to single exponential functions.

Figure 3:. 3-D plot of aligned and normalized single nucleotide incorporation experiment electropherograms.

Data is from the experiment described in Figure 2. Time is shown on the X axis, “Data Point” shown on the Y-axis represents the normalized migration time (relative to the internal standard), and the fractional area normalized to peak height is shown on the Z-axis. The normalized migration time was calculated by the analysis software to correct for the differences in migration time between capillaries. Fractional area was calculated in the analysis software to determine a scaling factor for each electropherogram.

Figure 4:. Validation of RNA separation and analysis by CE.

Scheme: The RNA substrate used in the kinetics assay is shown, consisting of a 20 nt, 5′-[6-FAM]-labeled primer annealed to a 40 nt template. UTP and Remdesivir triphosphate were added and the extended RNA bases are shown in red. Experimental conditions: A solution of 1.5 μM SARS CoV-2 RdRp (NSP12/7/8 plus 6 μM NSP8), 100 nM FAM-RNA, and 5 mM Mg²⁺ was mixed with 150 μM UTP and 40 μM Remdesivir-triphosphate to start the reaction in the quench flow at 37°C. Time points were quenched by mixing with EDTA from the quench syringe to 0.3 M. A) Sample electropherogram for a 0.325 second time point. Samples were injected for 6 seconds at 3.6 kV. Blue peaks correspond to FAM-labeled RNA products, while the yellow peak corresponds to the Cy3 internal standard. B) Concentration of RNA versus time. Data for different species were determined using the analysis software and are colored as listed in the top of the panel. Data are shown fit to a double exponentials function.