Nanoliter-scale sample preparation methods directly coupled to polymethylmethacrylate-based microchips and gel-filled capillaries for the analysis ofoligonucleotides

We are currently developing miniaturized, chip-based electrophoresis device s fabricated in plastics for the high-speed separation of oligonucleotides. One of the principal advantages associated with these devices is their sma ll sample requirements, typically in the nanoliter to sub-nanoliter range. Unfortunately, most standard sample preparation protocols, especially for o ligonucleotides, are done off-chip on a microliter-scale. Our work has focu sed on the development of capillary nanoreactors coupled to micro-separatio n platforms, such as micro-electrophoresis chips, for the preparation of se quencing ladders and also polymerase chain reactions (PCRs). These nanoreac tors consist of fused-silica capillary tubes (10-20 cmX20-50 mu m I.D.) wit h fluid pumping accomplished using the electroosmotic flow generated by the tubes. These reactors were situated in fast thermal cyclers to perform cyc le sequencing or PCR amplification of the DNAs. The reactors could be inter faced to either a micro-electrophoresis chips via capillary connectors micr omachined in polymethylmethacrylate (PMMA) using deep X-ray etching (width 50 mu m; depth 50 mu m) or conventional capillary gel tubes using zero-dead volume glass unions. For our chips, they also contained an injector, separ ation channel (length 6 cm; width 30 mu m; depth 50 mu m) and a dual fiber optic, near-infrared fluorescence detector. The sequencing nanoreactor used surface immobilized templates attached to the wall via a biotin-streptavid in-biotin linkage. Sequencing tracks could be directly injected into gel-fi lled capillary tubes with minimal degradation in the efficiency of the sepa ration process. The nanoreactor could also be configured to perform PCR rea ctions by filling the capillary tube with the PCR reagents and template. Af ter thermal cycling, the PCR cocktail could be pooled from multiple reactor s and loaded onto a slab gel or injected into a capillary tube or microchip device for fractionation. (C) 1999 Published by Elsevier Science B.V. All rights reserved.