ssDNA degradation along capillary electrophoresis process using a Tris buffer

Abstract

Tris-Acetate buffer is currently used in the selection and the characterization of ssDNA by capillary electrophoresis (CE). By applying high voltage, the migration of ionic species into the capillary generates a current that induces water electrolysis. This phenomenon is followed by the modification of the pH and the production of Tris derivatives. By injecting ten times by capillary electrophoresis ssDNA (50 nM), the whole oligonucleotide was degraded. In this paper, we will show that the Tris buffer in the running vials is modified along the electrophoretic process by electrochemical reactions. We also observed that the composition of the metal ions changes in the running buffer vials. This phenomenon, never described in CE, is important for fluorescent ssDNA analysis using Tris buffer. The oligonucleotides are degraded by electrochemically synthesized species (present in the running Tris vials) until it disappears, even if the separation buffer in the capillary is clean. To address these issues, we propose to use a sodium phosphate buffer that we demonstrate to be electrochemically inactive.

An Ion Concentration Polarization Microplatform for Efficient Enrichment and Analysis of ctDNA

“…This may be attributed to three factors: (1) An excessive amount of EP produced by an excessive field strength (EP > EO); (2) An electro-kinetic instability induced by the liquid vortex under high voltage, which results in extra ion transport and overlimited current; 29 (3) FAM-ssDNA degradation caused by the pH change of the solution and the impact of electrochemical free radicals (like metal ions in the electrode), which hindered the establishment of the steady enrichment effect. 30 Considering its acceptable rate and enrichment efficiency, 100 V was fixed in our following experiments. Next, we calculated the fluorescence intensity of the biochip in the region of interest (ROI) every 10 s and found that DNA reached the maximum enrichment within 30 s, with the fluorescence peak at 600−1200 μm above the Nafion membrane (Figure 2i,j).…”

“…When the voltage was >100 V, even though a stronger fluorescence peak could be attained, it seemed to be fleeting. This may be attributed to three factors: (1) An excessive amount of EP produced by an excessive field strength ( EP > EO ); (2) An electro-kinetic instability induced by the liquid vortex under high voltage, which results in extra ion transport and overlimited current; (3) FAM-ssDNA degradation caused by the pH change of the solution and the impact of electrochemical free radicals (like metal ions in the electrode), which hindered the establishment of the steady enrichment effect . Considering its acceptable rate and enrichment efficiency, 100 V was fixed in our following experiments.…”

Nucleic acid degradation as barrier to gene delivery: a guide to understand and overcome nuclease activity

“…372 However, ssDNA has found to be degraded in Tris buffer during the CE measurements, indicating the importance of the buffer for CE. 373 To improve the sensitivity of the detection mode, Qin et al . developed a microchip method with laser induced fluorescence detection (MCE-LIF) to study the nuclease activity.…”

“…372 However, ssDNA has found to be degraded in Tris buffer during the CE measurements, indicating the importance of the buffer for CE. 373 To improve the sensitivity of the detection mode, Qin et al developed a microchip method with laser induced fluorescence detection (MCE-LIF) to study the nuclease activity. FAMlabeled ssDNA was degraded into 5-FAM-nucleoside monophosphates with greatly increased fluorescence intensity, and short non-labeled oligonucleotide fragments.…”