Evaluation of Microchip Electrophoresis as a Molecular Diagnostic Method for Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD), a genetic disease caused by mutations in the X chromosome, is currently diagnosed by Southern blot analysis. Affected males and female carriers are identified by the detection of duplicated or deleted exons in the dystropin gene. Disadvantages of this method include the large number of fragments required for hybridization to determine genomic alterations and the time-consuming, expensive, and tedious nature of the analysis. The deletions/duplications seen in DMD tend to be located at certain “hot spot” regions of the gene, however, allowing easy detection of a great number of cases by interrogating a limited number of PCR-amplified DNA fragments. Chamberlain and coworkers (1)(2) developed primers to amplify nine fragments from known deletion sites in a single multiplex PCR amplification. Beggs et al. (3) defined additional primers that amplify nine fragments in a second multiplex PCR. This total set of 18 fragments allows >97% of the deletions detectable by Southern blot analysis to be detected rapidly and accurately by PCR. Analysis of the PCR fragments is most frequently carried out on slab gels, but both the presence and amount of the correctly sized fragments are important because carriers have both the mutated and wild-type gene. Many clinical laboratories that offer DMD testing do not provide carrier testing because of the technical difficulties associated with quantitative analysis. To speed analysis and automate quantification, several groups have exploited capillary electrophoresis (CE) for the separation of DMD-relevant fragments (4)(5)(6). One of the groups that reported DMD detection by CE transferred their method to an electrophoretic microdevice (6), a very rapid separation technique demonstrated to be an effective tool for use in clinical analyses (7)(8)(9)(10)(11)(12)(13). Although these reports show CE and microchip electrophoresis …