Wall coating for DNA sequencing and fragment analysis by capillary electrophoresis

Abstract

In capillary electrophoresis, covering the inner capillary surface with a coating is an efficient way to minimize both the electroosmotic flow and sorption of analytes on the capillary wall. We modified the procedure by Cobb et al. [Anal. Chem. 62, 2478 (1990)] for preparing wall coating to permit large-scale production. Specifically, we use a positive pressure to fill the capillary with both thionyl chloride and later vinylmagnesium bromide solution. This enables large-scale production of the coating by treating 100 m capillary pieces at a time. We found that no extensive flushing with either organic solvents or sodium hydroxide is needed before the reactions are performed. Application of liquid thionyl chloride with positive pressure scavenges residual humidity on the capillary surface and eliminates a need for extensive drying of the capillary. In the polymerization step, elimination of TEMED from the polymerization mixture and incubation at 70°C enables a homogeneous coating to be prepared in capillaries as long as 100 m. The prepared wall coating is stable for approximately 110 runs of DNA sequencing in a denaturing matrix and over 300 runs of DNA fragment analysis under nondenaturing conditions. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 175–184, 1998