Optimization of Excitation and Detection Geometry for Multiplexed Capillary Array Electrophoresis of DNA Fragments

Abstract

A new on-column excitation and detection geometry for multiplexed array capillary electrophoresis is evaluated. The detection limit is improved 100-fold for the same laser power because of more efficient coupling of the laser beam. The illumination is also more uniform over the entire array. Stray light and cross-talk are effectively suppressed by immersing the capillaries in water to roughly match the refractive index. A charge-coupled-device (CCD) camera combined with cutoff filters provides adequate sensitivity and speed for high-speed DNA sequencing based on sieving in a polymer-matrix solution.

A fully automated multicapillary electrophoresis device for DNA analysis

“…The possible regeneration of capillaries enhances their lifetime. The sensitivity of 1´10 ±11 M (S/N = 40), which can be extrapolated to a detection limit in the very low pM range at S/N = 2, is comparable to that achieved with fiber optic detection (3.7´10 ±12 M [14]), confocal scanning systems (2´10 ±12 M [27]), and to that achieved with camera detection and side illumination [20]. Only sheath flow systems were reported to have a lower detection limit (1´10 ±13 M [5]).…”

“…Axial illumination has been described [4], but a better possibility is to couple the light from the side into the capillary array [11,14,20], similar to the illumination principle used in some slab gel sequencers. However, this requires a carefully focused and adjusted laser beam.…”

A capillary-array electrophoresis system using side-entry on-column laser irradiation combined with glass rod lenses

“…We [15] and other groups [14,16] have developed capillary-array systems that use both side-entry laser irradiation and on-column detection. In the original configuration of our system, all 24 capillaries were placed in air.…”

Miniaturized Injection Method Using Silver‐Coated Capillaries in DNA Sequencing by Multiplexed Capillary Electrophoresis

“…In all of the reports on multiplexed capillary electrophoresis for DNA sequencing [1,4,6], sample injection is carried out electrokinetically since gel-filled capillaries are employed to separate DNA fragments. Typically, the sample was injected from one vial to many (bundled) capillaries for evaluation.…”