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Central European Journal of Medicine

, Volume 9, Issue 6, pp 839–848 | Cite as

Sensitive quantification of mitochondrial mutation using new Taqman probes

  • Hue Thi Truong
  • Van-Anh Thi Nguyen
  • Hong-Loan Thi Nguyen
  • Van-Anh Pham
  • Tuan-Nghia Phan
Research Article
  • 138 Downloads

Abstract

The A3243G mitochondrial mutation is the major cause of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). The severity of the disease is correlated with the heteroplasmy level of the mutation. Here we describe for the first time the validation of a real-time polymerase chain reaction (PCR) assay with Taqman locked nucleic acid (LNA) fluorescent (FAM for mutant, HEX for wild type) probes for quantification of heteroplasmy levels in a total of 18 family members from 5 Vietnamese MELAS patients carrying A3243G. Almost no background of FAM signals was detected in normal samples, indicating that the probes were allele-specific. Standard curves indicate sensitive detection at 0.1% mutants and high reliability with R2 > 0.985. The correlation line between measured % mutant and expected % mutant was highly reliable, with a slope of 0.993 and R2 of 0.998. All positive A3243G mutant samples pre-screened by PCR-restriction fragment length polymorphism (RFLP) were confirmed, and their heteroplasmy levels quantified to be from 3.68 to 80.85%. The heteroplasmy levels in patients were higher than in their family members and generally correlated well with the severity of their clinical symptoms. Overall, this work is the first demonstration of the application of LNA probes for sensitive and highly reliable quantification of heteroplasmy levels in human mitochondria.

Keywords

A3243G mutation Mitochondrial DNA MELAS syndrome Real-time PCR Locked nucleic acid probe 

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Copyright information

© Versita Warsaw and Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Hue Thi Truong
    • 1
  • Van-Anh Thi Nguyen
    • 1
  • Hong-Loan Thi Nguyen
    • 1
  • Van-Anh Pham
    • 2
  • Tuan-Nghia Phan
    • 1
  1. 1.Key Laboratory of Enzyme and Protein TechnologyVNU University of ScienceThanhxuan, HanoiVietnam
  2. 2.Department of NeurologyNational Hospital for PediatricsDongda, HanoiVietnam

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