ProtocolsQualitative and quantitative detection of mitochondrial heteroplasmy in cerebrospinal fluid using denaturing high-performance liquid chromatography
Section snippets
Type of research
There is increasing evidence of the presence and extent to which mitochondrial heteroplasmy plays a role in disease susceptibility, particularly in tissues with high energy requirements. Recent studies demonstrate that accumulation of mitochondrial DNA (mtDNA) mutations accumulate with increasing age [3] and that the level of heteroplasmy is associated with age of onset and progression of chronic conditions, such as diabetic complications [1]. Studies to date have identified the level of
Time required
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mtDNA extraction: 1.5 h
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PCR of mitochondrial polymorphism: 3 h
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Gel electrophoresis of PCR products: 5 h
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dHPLC quantitation per PCR product: 15 min
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PCR product titrations: 0.5 h
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dHPLC analysis per sample: 14 min
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Entire procedure per sample for this particular polymorphism: ∼10.5 h.
Special equipment
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MJ Research PTC-0200 thermocycler (MJ Research, Waltham, MA)
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Gel electrophoresis apparatus (Owl Separation Systems, Portsmouth, NH)
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Gel documentation system (Kodak DC-120, New Haven, CT)
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Barnstead Easypure UV water purification system
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HPLC apparatus designed to conduct denaturing dHPLC: the dual plate WAVE® instrument (Transgenomic San Jose, CA), which consists of a Hitachi D-7000 Interface and includes the L-7100 Pump, L-7250 Autosampler, L-7300 Oven, L-7400 UV Detector and the L-7480 Fluorescent
Mitochondrial DNA extraction
Fresh CSF samples (3 ml) were collected from patients with severe traumatic brain injury (Glasgow Coma Score ≤8, 16–75 years of age, excluding penetrating head injuries) 12 h post injury. The CSF was collected by passively evacuating preexisting CSF from the tubing of the ventriculostomy located in the lateral ventricles of the brain. The ventriculostomies were placed as standard of care. The stopcock was turned on to the ventriculostomy tube and fresh CSF filled the tube. The stopcock was then
Detection of heteroplasmy
Each heteroplasmy titration was run in triplicate with excellent reliable replication of the data. More than one homoduplex, in addition to the presence of a heteroduplex, gave two indicators of heteroplasmy in all titrations where at least 5% heteroplasmy was present. In the dilutions between 1% and 0.01% heteroplasmy, the presence of a heteroduplex was the best indicator of heteroplasmy (Fig. 2). Each heteroduplex has two peaks of equal height, which is to be expected due to the fact that
Discussion
Reliably detecting and quantifying mtDNA heteroplasmy using a technique that does not require a priori knowledge of the type of mtDNA change is important if we are to understand the full extent that the mitochondrial genome plays in disease susceptibility. The dHPLC technique accesses the entire DNA fragment for sequence variation and has proven successful for this purpose. Heteroduplex formation served as the best qualitative indicator of heteroplasmy. All titrations resulted in heteroduplex
Essential references
[4].
Quick procedure
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mtDNA extraction from CSF samples
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Amplification of mitochondrial polymorphism
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Quantitation of PCR products and making titrations
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dHPLC analysis of the titrated samples
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Using peak heights determine heteroplasmy ratio.
Acknowledgements
The authors would like to thank the Competitive Medical Research Fund of the University of Pittsburgh Medical Center for their support of this project.
References (4)
- et al.
Clinical features of diabetic patients with 0.01–0.1% heteroplasmy A3243G mutation in leukocyte mitochondrial DNA
Diabetes Res. Clin. Pract.
(2001) - M.-N.I.f.G. Medicine, Online Mendelian inheritance in man, OMIM (TM). In: Johns Hopkins University (Baltimore, MD) and...