Effect of buffer flow on DNA separation in a microfabricated electrophoresis system

Abstract

An adequate buffer reservoir is one essential component of an electrophoresis system, providing current carrying ions and maintaining constant pH. In a microfabricated DNA separation system with on-chip electrodes, the amount of buffer used is limited by the design of the device; the buffer continuity can be easily disturbed by the production of bubbles. Continuously flowing 1 x Tris-borate-EDTA (TBE) buffer over the electrodes at the cathodic end solves both problems. This flow increases the resolution for ssDNA primer separations (21 and 25 bases) to a maximum value of 1.4 within a distance of 1.2 cm, about four times higher than that without flow. Similar improvement has been achieved for dsDNA separation (20 bp ladder; BioRad) at a distance of only 0.4 cm, giving baseline resolution for bands from 20 to 240 bp. We have also investigated the effect of buffer concentration on resolution, and no similar improvement can be obtained by merely increasing the buffer concentration without flow.

Transverse imaging and simulation of dsDNA electrophoresis in microfabricated glass channels

“…However, there are other problems associated with cross‐linked polyacrylamide. For instance, we have previously observed a loss in resolution for dsDNA bands larger than 200 bp in relatively dense gels (10%T) 10 or dsDNA bands larger than 500 bp in less‐concentrated gels (5%T) 6. This reduction significantly limits the resolvable size range of dsDNA especially in microfabricated devices with pre‐loaded gels.…”

“…Cross‐linked polyacrylamide is particularly attractive mainly due to its relatively short required separation distances as compared with other matrices 6, 7. The resolution of these systems has been optimized by several means, including electrode‐compaction injection 8, on‐chip fabricated detectors 9, and the introduction of buffer flow 10. The resolving power has been improved to a comparable level as that achieved by linear polymer solutions but at distances considerably shorter 11.…”