A modified procedure for quantitative analysis of mtDNA, detecting mtDNA depletion

• Authors: A. Weglewska , J. Jakobkiewicz-Banecka , G. Wegrzyn
• Languages of publication: EN

Abstracts

EN

Quantitative analysis of mitochondrial DNA (mtDNA) is crucial for proper diagnosis of diseases that are caused by or associated with mtDNA depletion. However, such a quantitative characterization of mtDNA is not a simple procedure and requires several laboratory steps at which potential errors can accumulate. Here, we describe a modified procedure for quantitative human mtDNA analysis. The procedure is based on using two PCR-amplified, fluorescein-labeled DNA probes, complementary to mtDNA (detection probe) and chromosomal 18S rDNA (reference probe), both of similar length. Thus, equal amounts of these probes can be used and, contrary to previously published procedures, no mtDNA purification (apart from total DNA isolation) or 18S rDNA cloning is necessary for probe preparation. Two separate hybridizations (each with one probe) are suggested instead of one hybridization with both probes; this decreases background signals and enables adjustment of the strength of specific signals from both probes, which is useful in the subsequent densitometric analysis after superimposing of both pictures. Using different DNA amounts for reactions, we have proved that the procedure is quantitative in a broad range of sample DNA concentrations. Moreover, we were able to detect mtDNA depletion unambiguously in tissue samples from patients suffering from diseases caused by dysfunction of mtDNA.

Keywords

EN

DEPLETION; DNA ANALYSIS; HUMAN DISEASE; MITOCHONDRIAL DNA; SYNDROME

Discipline

• GENETICS: GENETICS

• Publisher: Institute of Plant Genetics, Polish Academy of Sciences
• Journal: Journal of Applied Genetics
• Year: 2005
• Volume: 46
• Issue: 4
• Pages: 423-428

Contributors

author

A. Weglewska

author

J. Jakobkiewicz-Banecka

author

G. Wegrzyn

• Document Type: ARTICLE
• Publication order reference: G. Wegrzyn, Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland