Multiplex fluorescence-based primer extension method for quantitative mutation analysis of mitochondrial DNA and its diagnostic application for Alzheimer's disease
- 1 August 1997
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 25 (15) , 3102-3109
- https://doi.org/10.1093/nar/25.15.3102
Abstract
A sensitive and highly reproducible multiplexed primer extension assay is described for quantitative mutation analysis of heterogeneous DNA populations. Wild-type and mutant target DNA are simultaneously probed in competitive primer extension reactions using fluorophor-labeled primers and high fidelity, thermostable DNA polymerases in the presence of defined mixtures of deoxy- and dideoxynucleotides. Primers are differentially extended and the resulting products are distinguished by size and dye label. Wild-type:mutant DNA ratios are determined from the fluorescence intensities associated with electrophoretically resolved reaction products. Multiple nucleotide sites can be simultaneously interrogated with uniquely labeled primers of different lengths. The application of this quantitative technique is shown in the analysis of heteroplasmic point mutations in mitochondrial DNA that are associated with Alzheimer's disease.Keywords
This publication has 33 references indexed in Scilit:
- RETRACTED: Mutations in mitochondrial cytochrome c oxidase genes segregate with late-onset Alzheimer diseaseProceedings of the National Academy of Sciences, 1997
- Mitochondrial disorders. Methods and specimen selection for diagnostic molecular pathology.1996
- Single nucleotide primer extension: quantitative range, variability, and multiplex analysis.Genome Research, 1996
- Longitudinal study of a heteroplasmic 3460 Leber hereditary optic neuropathy family by multiplexed primer-extension analysis and nucleotide sequencing.1996
- Mutation detection by solid phase primer extensionHuman Mutation, 1996
- A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.Proceedings of the National Academy of Sciences, 1995
- Quantitative determination of heteroplasmy in Leber's hereditary optic neuropathy by single-strand conformation polymorphism.1995
- Fluorescence-based DNA minisequence analysis for detection of known single-base changes in genomic DNAMolecular and Cellular Probes, 1995
- Novel use of limited primer extension in detecting mutations in human iduronate 2-sulfatase gene.1995
- Sequence and organization of the human mitochondrial genomeNature, 1981