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A rapid and accurate approach to identify single nucleotide polymorphisms of mitochondrial DNA using MALDI-TOF mass spectrometry
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Alex Xiu-Cheng Fan
Published/Copyright:
February 6, 2008
Abstract
Background: Single nucleotide polymorphisms (SNPs) of mitochondrial DNA (mtDNA) are involved in physiological and pathological conditions. We developed a rapid, accurate, highly sensitive and high-throughput approach with low cost to identify mtDNA SNPs.
Methods: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect 18 SNPs of mtDNA by uniplex and multiplex assays. The sensitivity and specificity of the MALDI-TOF MS were evaluated. The accuracy of the approach was validated by the comparison of using the robust sequencing analysis.
Results: The detection limit achieved with the assays corresponded to the identification of five-genome equivalence of mtDNA per reaction after first round PCR amplification. The testing system enabled the discrimination of as little as 5% of mtDNA polymorphism in the predominating background of mtDNA not containing the SNP. No false positive and false negative results were obtained using the uniplex and multiplex MALDI-TOF MS assays for the analysis of the 18 SNPs compared with those obtained by sequencing analysis.
Conclusions: Possible fields which could benefit from this powerful and sensitive tool include forensic medicine, tracing of matrilineage, transplantation immunology, transfusion medicine, the diagnosis of mtDNA mutation related disorders, and the research regarding aging, apoptosis and carcinogenesis based on physiologic and pathogenic alterations of mtDNA for the analysis of large-scale samples, multiple SNPs or rare mtDNA.
Clin Chem Lab Med 2008;46:299–305.
Keywords: high-throughput analysis; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); mitochondrial DNA (mtDNA); multiplex assay; single nucleotide polymorphisms (SNPs); uniplex assay
Received: 2007-10-2
Accepted: 2007-11-8
Published Online: 2008-02-6
Published in Print: 2008-03-01
©2008 by Walter de Gruyter Berlin New York
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- Size distribution of circulating cell-free DNA in sera of breast cancer patients in the course of adjuvant chemotherapy
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