Molecular diagnostics on microfabricated electrophoretic devices: From slab gel- to capillary- to microchip-based assays for T- and B-cell lymphoproliferative disorders

Background: Current methods for molecular-based diagnosis of disease rely h eavily on modern molecular biology techniques for interrogating the genome for aberrant DNA sequences. These techniques typically include amplificatio n of the target DNA sequences followed by separation of the amplified fragm ents by slab gel electrophoresis. As a result of the labor-intensive, time- consuming nature of slab gel electrophoresis, alternative electrophoretic f ormats have been developed in the form of capillary electrophoresis and, mo re recently, multichannel microchip electrophoresis. Methods: Capillary electrophoresis was explored as an alternative to slab g el electrophoresis for the analysis of PCR-amplified products indicative of T- and B-cell malignancies as a means of defining the elements for silica microchip-based diagnosis. Capillary-based separations were replicated on e lectrophoretic microchips. Results: The microchip-based electrophoretic separation effectively resolve d PCR-amplified fragments from the variable region of the T-cell receptor-g amma gene (150-250 bp range) and the immunoglobulin heavy chain gene (80-14 0 bp range), yielding diagnostically relevant information regarding the pre sence of clonal DNA populations. Although hydroxyethylcellulose provided ad equate separation power, the need for a coated microchannel for effective r esolution necessitated additional preparative steps. In addition, prelimina ry data are shown indicating that polyvinylpyrrolidone may provide an adequ ate matrix without the need for microchannel coating. Conclusions: Separation of B- and T-cell gene rearrangement PCR products on microchips provides diagnostic information in dramatically reduced time (1 60 s vs 2.5 h) with no loss of diagnostic capacity when compared with curre nt methodologies. As illustrated, this technology and methodology holds gre at potential for extrapolation to the abundance of similar molecular biolog y-based techniques. (C) 1999 American Association for Clinical Chemistry.