Integrated continuous flow polymerase chain reaction and micro-capillary electrophoresis system with bioaffinity preconcentration

Abstract

An integrated and modular DNA analysis system is reported that consists of two modules: (i) A continuous flow polymerase chain reaction (CFPCR) module fabricated in a high T g (150°C) polycarbonate substrate in which selected gene fragments were amplified using biotin and fluorescently labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones as opposed to heating and cooling large thermal masses typically performed in batch-type thermal reactors. (ii) μCE (micro-capillary electrophoresis) module fabricated in poly(methylmethacrylate) (PMMA), which utilized a bioaffinity selection and purification bed (2.9μL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to electrophoretic sorting. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed, where they were extracted onto the surface of micropillars. The affinity bed was also fabricated in PMMA and was populated with an array of microposts (50μm width; 100μm height) yielding a total surface area of ~117mm 2. This solid-phase extraction (SPE) process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (K d = 10 -15M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single-stranded DNA released for injection into a 7-cm-long μCE channel for size-based separations and fluorescence detection. The utility of the system was demonstrated using Alu DNA typing for gender and ethnicity determinations as a model. Compared with the traditional cross-T injection procedure typically used for μCE, the affinity pre-concentration and injection procedure generated signal enhancements of 17- to 40-fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation.