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

• Autorzy: Weglewska A , Jakobkiewicz-Bonecka J. , Wegrzyn G.
• Języki publikacji: EN

Abstrakty

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.

• Wydawca: -
• Czasopismo: Journal of Applied Genetics
• Rocznik: 2005
• Tom: 46
• Numer: 4
• Opis fizyczny: p.423-428,fig.,ref.

Twórcy

autor

Weglewska A

• Univerity of Gdansk, Kladki 24, 80-822 Gdansk, Poland

autor

Jakobkiewicz-Bonecka J.

autor

Wegrzyn G.

Bibliografia

• Arnaudo E, Dalakas M, Shanske S, Moraes CT, DiMauro S, Schon EA, 1991. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 337: 508-510.
• Barrientos A, Casademont J, Cardellach F, Estivill X, Urbano-Marquez A, Nunes V, 1997. Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging. Brain Res Mol Brain Res 52: 284-289.
• Bradley JL, Blake JC, Chamberlain S, Thomas PK, Cooper JM, Schapira AH, 2000. Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia. Hum Mol Genet 9: 275-282.
• Brown MD, Wallace DA, 1994. Molecular basis of mitochondrial DNA disease. J Bioenerg Biomembr 26: 273-289.
• Cantatore P, Petruzzella V, Nicoletti C, Papadia F, Fracasso F, Rustin P, et al. 1998. Alteration of mitochondrial DNA and RNA level in human fibroblasts with impaired vitamin B12 coenzyme synthesis. FEBS Lett 432: 173-178.
• Cazzalini O, Lazze MC, Iamele L, Stivala LA, Bianchi L, Vaghi P, et al. 2001. Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes. Biochem Pharmacol 62: 893-902.
• Elpeleg O, 2003. Inherited mitochondrial DNA depletion. Pediatr Res 54: 153-159.
• Elpeleg O, Mandel H, Saada A, 2002. Depletion of the other genome-mitochondrial DNA syndromes in humans. J Mol Med 80: 389-396.
• Kirches EJ, Winkler K, Warich-Kirches M, Szibor R, Wien F, Kunz WS, et al. 1998. mtDNA depletion and impairment of mitochondrial function in a case of a multisystem disorder including severe myopathy. I Inher Metab Dis 21: 400-408.
• Macmillan CJ, Shoubridge EA, 1996. Mitochondrial DNA depletion: prevalence in a pediatric population referred for neurologic evaluation. Pediatr Neurol 14: 203-210.
• Papadimitriou A, Comi GP, Hadjigeorgiou GM, Bordoni A, Sciacco M, Napoli L, et al. 1998. Partial depletion and multiple deletions of muscle mtDNA in familial MNGIE syndrome. Neurology 51: 1087-1092.
• Pesce V, Cormio A, Fracasso F, Vecchiet J, Felzani G, Lezza AM, et al. 2001. Age-related mitochondrial genotypie and phenotypic alterations in human skeletal muscle. Free Radie Biol Med 30: 1223-1233.
• Pesce V, Cormio A, Marangi LC, Guglielmi FW, Lezza AM, Francavilla A, et al. 2002. Depletion of mitochondrial DNA in the skeletal muscle of two cirrhotic patients with severe asthenia. Gene 286: 143-148.
• Pulkes T, Hanna MG, 2001. Human mitochondrial DNA diseases. Adv Drug Deliv Rev 49: 27-43.
• Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, Elpeleg O, 2001. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 29: 342-344.
• Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning: A Laboratory Mannual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
• Sciacco M, Gasparo-Rippa P, Vu TH, Tanji K, Shanske S, Mendell JR, et al. 1998. Study of mitochondrial DNA depletion in muscle by single-fiber polymerase chain reaction. Muscle Nerve 21: 1374-1381.
• Scotti C, Iamele L, Alessandrini A, Vannini V, Cazzalini O, Lazze Mc, et al. 2003. Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria. Mitochondrion 2: 361-373.
• Śrutkowska S, Caspi R, Gabig M, Węgrzyn G, 1999. Detection of DNA replication intermediates after two-dimensional agarose gel electrophoresis using a fluorescein-labeled probe. Anal Biochem 269: 221-222.
• Taanman JW, Bodnar AG, Cooper JM, Morris AA, Clayton PT, Leonard JV, et al. 1997. Molecular mechanisms in mitochondrial DNA depletion syndrome. Hum Mol Genet 6: 935-942.
• Wiedemann FR, Manfredi G, Mawrin C, Beal MF, Schon EA, 2002. Mitochondrial DNA and respiratory chain function in spinal cords of ALS patients. J Neurochem 80: 616-625.