Wall coating for DNA sequencing and fragment analysis by capillary electrophoresis

Abstract

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 w analytes on the capillary wall. We modified the procedure by Cobb et al. Anal.Ž .x 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 70ЊC 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.

Recent progress in DNA analysis by capillary electrophoresis

“…The confocal system utilizes a set of four photomultiplier tubes with proper filters and dichroic beam splitters. Columns coated with LPA and filled with low-viscosity LPA solutions are used [68]. The equipment from Perkin-Elmer employs postcolumn detection with liquid sheath flow [61,69].…”

Polymer wall coatings for capillary electrophoresis

“…To improve the stability of the final coating, Cobb and co-workers [61] replaced the Si-O-Si bonds with Si-C bonds in a procedure using Grignard chemistry. This procedure has been modified later to allow large-scale production of capillaries to produce coated capillary arrays for the MegaBA-CE TM DNA sequencer [62]. Nakatani and co-workers [63] modified the original Grignard chemistry method and confirmed the stability of the Si-C bond based coating.…”

Galactomannans as a sieving matrix in capillary electrophoresis

“…Electrophoresis was performed at room temperature in capillaries (75 mm ID, 200 mm OD) with a total length of 65 cm and an effective length of 40 cm coated with linear polyacrylamide [48,49]. The sieving matrix was prepared as described above.…”