The study describes the method of a sensitive detection of double-stranded DNA molecules in situ. It is based on the oxidative attack on the deoxyribose moiety by copper(I) in the presence of oxygen. We have shown previously that the oxidative attack leads to the formation of frequent gaps in DNA. Here we have demonstrated that the gaps can be utilized as the origins for an efficient synthesis of complementary labeled strands by DNA polymerase I and that such enzymatic detection of the double-stranded DNA is a sensitive approach enabling in-situ detection of both the nuclear and mitochondrial genomes in formaldehyde-fixed human cells.

Laboratory of Experimental Medicine Institute of Molecular and Translational Medicine Faculty of Medicine Palacký University in Olomouc Olomouc Czech Republic

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Colson P, Houssier C, Bailly C (1995) Use of electric linear dichroism and competition experiments with intercalating drugs to investigate the mode of binding of Hoechst 33258, berenil and DAPI to GC sequences. J Biomol Struct Dyn 13: 351–366. PubMed

Kapuscinski J (1995) DAPI: a DNA-specific fluorescent probe. Biotech Histochem 70: 220–233. PubMed

Hirons GT, Fawcett JJ, Crissman HA (1994) TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry. Cytometry 15: 129–140. PubMed

Rye HS, Yue S, Wemmer DE, Quesada MA, Haugland RP, et al. (1992) Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res 20: 2803–2812. PubMed PMC

Tanious FA, Veal JM, Buczak H, Ratmeyer LS, Wilson WD (1992) DAPI (4′,6-diamidino-2-phenylindole) binds differently to DNA and RNA: minor-groove binding at AT sites and intercalation at AU sites. Biochemistry 31: 3103–3112. PubMed

van de Corput MP, van den Ouweland JM, Dirks RW, Hart LM, Bruining GJ, et al. (1997) Detection of mitochondrial DNA deletions in human skin fibroblasts of patients with Pearson's syndrome by two-color fluorescence in situ hybridization. J Histochem Cytochem 45: 55–61. PubMed

Wang Y, Bogenhagen DF (2006) Human mitochondrial DNA nucleoids are linked to protein folding machinery and metabolic enzymes at the mitochondrial inner membrane. J Biol Chem 281: 25791–25802. PubMed

Ligasova A, Strunin D, Liboska R, Rosenberg I, Koberna K (2012) Atomic scissors: a new method of tracking the 5-bromo-2′-deoxyuridine-labeled DNA in situ. PLoS One 7: e52584. PubMed PMC

Magnusson J, Orth M, Lestienne P, Taanman JW (2003) Replication of mitochondrial DNA occurs throughout the mitochondria of cultured human cells. Exp Cell Res 289: 133–142. PubMed

Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105: 2415–2420. PubMed PMC

Davis AF, Clayton DA (1996) In situ localization of mitochondrial DNA replication in intact mammalian cells. J Cell Biol 135: 883–893. PubMed PMC

Haines KM, Feldman EL, Lentz SI (2010) Visualization of mitochondrial DNA replication in individual cells by EdU signal amplification. J Vis Exp. PubMed PMC

Iborra FJ, Kimura H, Cook PR (2004) The functional organization of mitochondrial genomes in human cells. BMC Biol 2: 9. PubMed PMC

Pogozelski WK, Tullius TD (1998) Oxidative Strand Scission of Nucleic Acids: Routes Initiated by Hydrogen Abstraction from the Sugar Moiety. Chem Rev 98: 1089–1108. PubMed

Liboska R, Ligasova A, Strunin D, Rosenberg I, Koberna K (2012) Most Anti-BrdU Antibodies React with 2′-Deoxy-5-Ethynyluridine - The Method for the Effective Suppression of This Cross-Reactivity. PLoS One 7: e51679. PubMed PMC

Ligasová A, Koberna K (2010) In situ reverse transcription: the magic of strength and anonymity. Nucleic Acids Res 38: e167. PubMed PMC

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