Quantification of the 4977-bp deletion in human mitochondrial DNA using real-time PCR

Schinogl, Pamela; Müller, Mathias; Steinborn, Ralf

doi:10.1016/s0379-0738(01)00473-x

Cited by 5 publications

References 4 publications

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Quantitative Analysis of Somatic Mitochondrial DNA Mutations by Single-Cell Single-Molecule PCR

Kraytsberg

Bodyak

Myerow

et al. 2009

Methods in Molecular Biology

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Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an ongoing mutation process. Single-molecule PCR (smPCR) has been shown to be an effective method that can be applied to all areas of quantitative studies. It has distinct advantages over conventional vector-based cloning techniques avoiding the well-known PCR-related artifacts such as the introduction of artificial mutations, preferential allelic amplifications, and "jumping" PCR. smPCR is a straightforward and robust method, which can be effectively used for molecule-by-molecule mutational analysis, even when mitochondrial whole genome (mtWG) analysis is involved. This chapter describes the key features of the smPCR method and provides three examples of its applications in single-cell analysis: di-plex smPCR for deletion quantification, smPCR cloning for clonal point mutation quantification, and smPCR cloning for whole genome sequencing (mtWGS).

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Quantitative Analysis of Somatic Mitochondrial DNA Mutations by Single-Cell Single-Molecule PCR

Kraytsberg

Bodyak

Myerow

et al. 2009

Methods in Molecular Biology

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Techniques and Pitfalls in the Detection of Pathogenic Mitochondrial DNA Mutations

Moraes¹,

Atencio²,

Oca‐Cossio³

et al. 2003

The Journal of Molecular Diagnostics

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Mutations in the mitochondrial DNA (mtDNA) are now recognized as major contributors to human pathologies and possibly to normal aging. A large number of rearrangements and point mutations in protein coding and tRNA genes have been identified in patients with mitochondrial disorders. In this review, we discuss genotype-phenotype correlations in mitochondrial diseases and common techniques used to identify pathogenic mtDNA mutations in human tissues. Although most of these approaches employ standard molecular biology tools, the co-existence of wild-type and mutated mtDNA (mtDNA heteroplasmy) in diseased tissues complicates both the detection and accurate determination of the size of the mutated fractions. To address these problems, novel approaches were developed and are discussed in this review.

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Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5′-nuclease (TaqMan™) real-time polymerase chain reaction assay and plasmid external calibration standards

Pogozelski

Hamel²,

Woeller

et al. 2003

Mitochondrion

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Quantification of the 4977-bp deletion in human mitochondrial DNA using real-time PCR

Cited by 5 publications

References 4 publications

Quantitative Analysis of Somatic Mitochondrial DNA Mutations by Single-Cell Single-Molecule PCR

Quantitative Analysis of Somatic Mitochondrial DNA Mutations by Single-Cell Single-Molecule PCR

Techniques and Pitfalls in the Detection of Pathogenic Mitochondrial DNA Mutations

Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5′-nuclease (TaqMan™) real-time polymerase chain reaction assay and plasmid external calibration standards

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