HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Boles, R. G. Articles by Ito, M. Search for Related Content PubMed PubMed Citation Articles by Boles, R. G. Articles by Ito, M. Related Collections Molecular Diagnostics and Genetics

Quantification of Mitochondrial DNA Heteroplasmy by Temporal Temperature Gradient Gel Electrophoresis

¹ Division of Medical Genetics, Childrens Hospital Los Angeles, Los Angeles, CA 90027

² Department of Pediatrics, Keck School of Medicine at the University of Southern California, Los Angeles, CA 90033

³ Colibri Pro Development, S-187 76 Täby, Sweden

^aaddress correspondence to this author at: Division of Medical Genetics, Box 90, Childrens Hospital Los Angeles, Los Angeles, CA 90027; fax 323-665-5937, e-mail rboles@chla.usc.edu

The first 300 words of the full text of this article appear below.

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 . . . [Full Text of this Article]