Rapid Analysis of Mitochondrial DNA Heteroplasmy in Diabetes by Gel-Microchip Electrophoresis

Abstract

Gel-microchip electrophoresis is a novel combination of the well-established methods of slab-gel electrophoresis and capillary-gel electrophoresis (1). The gel-microchip format provides a multilane separation platform (a plurality of virtual channels up to 96 lanes) with excellent heat dissipation characteristics, allowing application of high voltages necessary to obtain rapid and high-performance analysis of DNA fragments. The system readily accommodates fluorescent labeling during the electrophoresis separation process (in migratio), such as intercalation with ethidium bromide or complexation with other novel, high-sensitivity DNA staining dyes, in addition to the use of conventional covalently labeled primers (i.e., before the separation process). Sample injection onto the gel microchip is accomplished by membrane-mediated loading technology (2), which also enables robotic spotting of multiple samples. The method is readily applicable for large-scale, automated, high-throughput mutation screening (3). Mutations in mitochondrial DNA (mtDNA) (4) are recognized as important in diseases ranging from ischemic heart disease to neurodegenerative disorders (5) and diabetes (6)(7)(8)(9)(10). mtDNA mutations accumulate with age and may play an important role in aging (11). The association of diabetes with the mtDNA tRNA Leu (UUR) A3243G point mutation responsible for the syndrome of mitochondrial encephalomyopathy with ragged red fibers and stroke-like episodes (MELAS) was reported in 1992 in a family with maternally inherited deafness and type II diabetes (7). Differences in the prevalence of the MELAS mutation among ethnic groups (9)(10)(12) highlighted the need for accurate, high-sensitivity screening for the MELAS 3243 and other tRNA mutations. Most studies searching for the 3243 mutation in diabetes have focused on non-insulin-dependent diabetes mellitus; however, many MELAS patients are insulin dependent (9)(10), a fact that suggests that all types of diabetic patients, and particularly type II patients, should be tested if the true incidence …