Screening for human mitochondrial DNA polymorphisms with denaturing gradient gel electrophoresis

Abstract

A method involving denaturing gradient gel electrophoresis (DGGE) was developed to detect mitochondrial DNA (mtDNA) polymorphisms in human peripheral T-lymphocytes. DGGE analysis of 100- to 200-bp sequences of low melting temperature domains within the origin/membrane attachment site, NADH dehydrogenase subunit I, cytochrome c oxidase subunit I and two overlapping regions of the tRNA glycine/NADH dehydrogenase subunit III sequences was performed to identify sequence variants at these sites in a human B-cell line TK6 and T-cells from four individuals. A T --> C transition at position 16519 in the origin/membrane attachment site in the TK6 cell line and the T-cells from one individual was found. A sequence variant resulting in a G --> A transition at position 9966 in the tRNA glycine/NADH dehydrogenase III was identified in another individual. This method should be useful for the rapid screening of polymorphisms in a large number of samples.

DNA repair of UV photoproducts and mutagenesis in human mitochondrial DNA

“…As already described for other cell lines (Hanekamp et al, 1996), we detected a polymorphism within the mt fragment of 293 cells analysed, namely an AT > GC transition at position 3197. This ®nding con®rms that mutations can accumulate in the mt genome during serial passages of established cell lines.…”

Exhaustive Scanning Approach to Screen All the Mitochondrial tRNA Genes for Mutations and Its Application to the Investigation of 35 Independent Patients with Mitochondrial Disorders

“…First, such mutations may occur at any position, thereby underlining the need to develop exhaustive screening methods; second, after detecting and identifying new sequence variations, classifying them as polymorphisms or pathogenic mutations may be difficult: one needs especially to know if they are present in a homoplasmic or heteroplasmic state. Among the different screening techniques that have been proposed to study mtDNA (26)(27)(28)(29)(30)(31)(32), a DGGEbased approach using a GC-clamp and heteroduplex detection is a powerful way to overcome these two difficulties. Indeed, it relies on a screening method that is exhaustive in optimal experimental conditions (10,33); in addition, it allows direct detection of a minor heteroplasmy that can escape standard molecular analyses based on direct sequencing or on the cloning of PCR-amplified fragments.…”

A Sensitive Denaturing Gradient-Gel Electrophoresis Assay Reveals a High Frequency of Heteroplasmy in Hypervariable Region 1 of the Human mtDNA Control Region**Disclaimer: The opinions and assertions expressed herein are solely those of the authors and are not to be construed as official or the views of the United States Department of Defense, the United States Department of the Army, or the United States Department of Commerce. This paper is a contribution of the United States National Institute of Standa

“…We have implemented a denaturing gradient-gel electrophoresis (DGGE) assay, to perform a population survey of heteroplasmy in the HV1 portion of the mtDNA CR. DGGE has been successfully used to identify mutant sequences in nuclear genes (Traystman et al 1990;Laubscher et al 1994) as well as heteroplasmy in the mitochondrial genome (Yoon et al 1991;Lombes et al 1992;Hanekamp et al 1996;Jazin et al 1996;Michikawa et al 1997;Danan et al 1999;Michikawa et al 1999). In the present study, we report both the frequency of HV1 heteroplasmy in the population, on the basis of a survey of blood samples from 253 individuals, and estimates of the heteroplasmic ratios present in those individuals determined to be heteroplasmic.…”