Stability of capillary gels for automated sequencing of DNA

Abstract

Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of a resistive region at the anode end in DNA capillary electrophoresis

“…Early observations of the anomalous behavior during the capillary DNA separation were reported in cross-linked gels. The effect was often described as a decrease in the capillary current, "loss of gel stability" and "gel breakdown" [5][6][7]. Since the cross-linked gels were intended for multiple use, the authors resorted to clipping the cathode end of the capillary to restore the sequencing ability of the capillary [5,8].…”

Capillary electrophoresis in sieving matrices: Selectivity per base, mobility slope, and inflection slope

“…Capillary gel electrophoresis enables high-concentration gels (over 20% polyacrylamide) to be used for analysis of difficult-to-separate compounds such as polyamino acids [4,5]. However, the usefulness of cross-linked polymers in CE is restricted by their limited lifetime [6]. This has led to the introduction of solutions of linear polymers as sieving media in capillary electrophoresis [7,8].…”

DNA sequencing by capillary array electrophoresis and microfabricated array systems

“…However, CGE still had some features that were not compatible with a high-throughput regime. Among them were the short column lifetime as well as injection-related problems [29,30].…”