Quantification of mitochondrial DNA heteroplasmy by temporal temperature gradient gel electrophoresis.

Abstract

Mutations of mitochondrial DNA are increasingly becoming recognized as causes of a wide variety of disorders, most notably neuromuscular and multisystem. In the vast majority of disease-causing mitochondrial DNA (mtDNA) mutations reported to date, mutated and wild-type DNA coexist in the same cell, a condition referred to as “heteroplasmy” (1). Different tissues can contain various proportions of mutant and wild-type species, and although far from a perfect correlation, determination of these proportions in accessible tissues has some clinical relevance (1) and is often performed by molecular diagnostic laboratories. Because >100 disease-associated sequence changes have been reported in virtually every region of the mtDNA (2), rapid heteroplasmy screening assays have been used by some laboratories to detect both rare and novel mutations. One such assay, temporal temperature gradient gel electrophoresis (TTGE), is sensitive and specific for mtDNA heteroplasmy. In addition, each heteroplasmic variation produces a distinctive band pattern (3). In TTGE, whole cellular DNA is isolated from tissue and the segment of interest is PCR-amplified. Gel electrophoresis of amplified products occurs with the gel immersed in buffer solution, and the entire gel slowly and methodically warms through the melting temperature range particular to the specific DNA segment, allowing the separation of DNA fragments based on minute sequence differences. At the conclusion of the run, gels are bathed in ethidium bromide and photographed by a charge-coupled device digital camera system (3)(4). In the presence of a single mtDNA species (homoplasmy), a single gel band results. If heteroplasmy is present, melting and reannealing after the last PCR cycle produces two homoduplex and two heteroduplex double-stranded species with different mobilities at approximately their melting temperature, corresponding to the presence of multiple gel bands (3)(4). Although in theory four bands should occur in the presence of heteroplasmy, in practice the …