Landers, James P.

Abstract Background: Current methods for molecular-based diagnosis of disease rely heavily on modern molecular biology techniques for interrogating the genome for aberrant DNA sequences. These techniques typically include amplification of the target DNA sequences followed by separation of the amplified fragments by slab gel electrophoresis. As a result of the labor-intensive, time-consuming nature of slab gel electrophoresis, alternative electrophoretic formats have been developed in the form of capillary electrophoresis and, more recently, multichannel microchip electrophoresis. Methods: Capillary electrophoresis was explored as an alternative to slab gel 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 electrophoretic microchips. Results: The microchip-based electrophoretic separation effectively resolved PCR-amplified fragments from the variable region of the T-cell receptor-γ gene (150–250 bp range) and the immunoglobulin heavy chain gene (80–140 bp range), yielding diagnostically relevant information regarding the presence of clonal DNA populations. Although hydroxyethylcellulose provided adequate separation power, the need for a coated microchannel for effective resolution necessitated additional preparative steps. In addition, preliminary data are shown indicating that polyvinylpyrrolidone may provide an adequate 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 (160 s vs 2.5 h) with no loss of diagnostic capacity when compared with current methodologies. As illustrated, this technology and methodology holds great potential for extrapolation to the abundance of similar molecular biology-based techniques.

Abstract Capillary electrophoresis is a relatively new analytical technique that has begun to have an impact in the clinical laboratory, both for routine analyses and for those that are more esoteric. Its potential for automated, rapid, high-efficiency separations makes it appealing as a replacement for some of the more labor-intensive assays carried out in electrophoretic gels and as a complement to companion techniques such as HPLC. Among the many attractive characteristics of this technology is its versatility for analyses of a diverse spectrum of analytes, ranging from small organic ions to macromolecular protein complexes or DNA. The focus of this commentary is to familiarize the clinical scientist with the instrumentation and principles of capillary electrophoretic separation and to review the recent research demonstrating the applicability of this technology to the clinical laboratory.