Rapid identification of mitochondrial DNA (mtDNA) mutations in neuromuscular disorders by using surveyor strategy
Department of Medical Genetics, Archet 2 Hospital, CHU Nice, France. Mitochondrion
(Impact Factor: 3.25).
04/2008; 8(2):136-45. DOI: 10.1016/j.mito.2007.10.008
Mutations of mitochondrial genome are responsible for respiratory chain defects in numerous patients. We have used a strategy, based on the use of a mismatch-specific DNA endonuclease named " Surveyor Nuclease", for screening the entire mtDNA in a group of 50 patients with neuromuscular features, suggesting a respiratory chain dysfunction. We identified mtDNA mutations in 20% of patients (10/50). Among the identified mutations, four are not found in any mitochondrial database and have not been reported previously. We also confirm that mtDNA polymorphisms are frequently found in a heteroplasmic state (15 different polymorphisms were identified among which five were novel).
Available from: Hsiu-Chuan Yen
- "Janne et al. has applied this platform for high-sensitivity mutation screening of the epidermal growth factor receptor (EGRF) gene in human cancer specimens , but it has not been applied for the detection of mtDNA mutation. Although Bannwarth et al. have developed 17 primer pairs for screening mutations of entire human mtDNA by using SN analysis, they detected cleaved DNA fragments by using agarose gel electrophoresis , . Because SN/WAVE-HS analysis does not require the testing on optimal temperatures and can be used to analyze amplicons much longer than 500 bp, it is very likely to be a more superior technique than DHPLC for the efficient screening of unknown heteroplasmic variants or mutations in entire mtDNA. "
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ABSTRACT: High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detecting human mtDNA variants and found that its performance was slightly better than that of denaturing high-performance liquid chromatography (DHPLC). The potential interference from co-amplified NUMTs on screening mtDNA heteroplasmy when using these 2 highly sensitive techniques was further examined by using 2 published primer sets containing a total of 65 primer pairs, which were originally designed to be used with one of the 2 techniques. We confirmed that 24 primer pairs could amplify NUMTs by conducting bioinformatic analysis and PCR with the DNA from 143B-ρ0 cells. Using mtDNA extracted from the mitochondria of human 143B cells and a cybrid line with the nuclear background of 143B-ρ0 cells, we demonstrated that NUMTs could affect the patterns of chromatograms for cell DNA during SN-WAVE/HS analysis of mtDNA, leading to incorrect judgment of mtDNA homoplasmy or heteroplasmy status. However, we observed such interference only in 2 of 24 primer pairs selected, and did not observe such effects during DHPLC analysis. These results indicate that NUMTs can affect the screening of low-level mtDNA variants, but it might not be predicted by bioinformatic analysis or the amplification of DNA from 143B-ρ0 cells. Therefore, using purified mtDNA from cultured cells with proven purity to evaluate the effects of NUMTs from a primer pair on mtDNA detection by using PCR-based high-sensitivity methods prior to the use of a primer pair in real studies would be a more practical strategy.
PLoS ONE 03/2014; 9(3):e92817. DOI:10.1371/journal.pone.0092817 · 3.23 Impact Factor
Available from: Cecil Thomas
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ABSTRACT: A third-generation multichannel data acquisition system has been designed for cardiac electrogram analysis. The system is capable of recording data from 240 electrode sites, sampling at 2000 Hz. The design required special-purpose front-end electronics for intraoperative recordings, and high-bandwidth data acquisition for analysis of data on line. These design requirements are an attempt to simplify the problems associated with patient safety (leakage current) and the rapid analysis of large amounts of data.< >
Engineering in Medicine and Biology Society, 1988. Proceedings of the Annual International Conference of the IEEE; 12/1988
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ABSTRACT: This chapter discusses two high sensitivity mutation detection methods to identify genetic variation: chemical and enzymatic cleavage of mismatch. Although there are several methods for high sensitivity detection of known mutations, unknown mutations are more difficult to uncover. However, methods for the latter have improved significantly and are utilized in practical applications. These include the establishment of a single nucleotide polymorphism (SNP) map for a specific part of the genome in a specific animal population or the screening for unknown mutations in important genes, such as cancer susceptibility genes. These genes are large, with many exons, and thus hundreds of possible mutations that affect the functions of those proteins are found. The mutation detection methods serve as tools for reverse genetics to screen for chemically induced point mutations in specific regions of specific genes and for specific mutations in the genome of emerging pathogenic microorganisms. In mutation screening methods, the mutated DNA helix is first converted to mismatch heteroduplexes when the polymerase chain reaction products of two alleles of the gene of interest are amplified, mixed, denatured, and rehybridized. Next, the chemical or enzymatic properties of a mismatched base are exploited to lead to a break in the DNA strand near the mismatch. Finally, a suitable fragment analysis method is used to visualize the shortened DNA fragments that are produced by the DNA break in either single-stranded or double-stranded form.
ChemInform 05/2007; 38(21). DOI:10.1002/chin.200721275
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