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. 2013 Jun 18;8(6):e66864.
doi: 10.1371/journal.pone.0066864. Print 2013.

A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA

Anna Ligasová  1 , Dmytro Strunin, Karel Koberna
Affiliations

A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA

Anna Ligasová et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The scheme of the method.
A simplified scheme of two basic alternatives of labeling cellular double-stranded DNA is shown. The common steps (fixation, permeabilization and copper(I)-mediated DNA cleavage leading to the gap formation) are followed by the labeling of DNA by means of DNA polymerase I or TdT. In the case of TdT, it is necessary to use the pre-incubation step with SAP in order to reconstitute the hydroxyl groups at the 3′ ends of the gaps. P and OH designate phosphate and hydroxyl groups at the 3′ ends of the gaps, respectively.
Figure 2
Figure 2. The detection of nuclear DNA in HeLa cells.
The detection of nuclear DNA by DNA polymerase I (A, D) or TdT (B, C, E) and Alexa 555-dUTP after 10-minute incubation in the cleavage solution is shown. In the case of TdT, the incubation with TdT was (B) or was not (C) preceded by SAP treatment to remove the phosphate groups from the 3′ ends. The cells were (A, B, C) or were not (D, E) incubated in the cleavage solution for 10 minutes before the enzymatic detection of DNA. The relative mean signal intensities and the standard deviations are shown in (F) for all these experiments. Barr: 20 µm.
Figure 3
Figure 3. The detection of overall DNA by various marker nucleosides.
The detection of overall DNA by DNA polymerase I and either by Alexa 555-dUTP (A, C, J, L; red in the color images) or biotin-dUTP (D, F, M, O; red in the color images) or BrdUTP (P, R; red in the color images) in HeLa cells after 10-minute incubation in the cleavage solution is shown. The cells were simultaneously stained by DAPI (B, C, E, F, H, I, K, L, N, O, Q, R; blue in the color images). The cells in A–F were incubated in the DNA polymerase I mixture containing dTTP. The cells in J–R were incubated in the DNA polymerase I mixture without the addition of dTTP. The cells in G–I were fixed, permeabilized, incubated with anti-BrdU antibody and secondary antibody, and the cells were stained by DAPI. Bar: 50 µm.
Figure 4
Figure 4. The detection of the mitochondrial genome.
The detection of the mitochondrial DNA by a 10-second cleavage in a solution containing copper(I) ions followed by the labeling of mitochondrial DNA by DNA polymerase I and biotin-dUTP (A, D; red in the color image) or Alexa 555-dUTP (F, I; red in the color image). The anti-mitochondrial antibody (B, D, G, I; green in the color images) was used for the identification of mitochondria. DNA was stained by DAPI (C, D, H, I; blue in the color images). The graphs show the relative mean signal intensities and the standard deviations of Alexa 555-dUTP (E) and biotin-dUTP (J) derived signal measured in the nucleus, mitochondria and cytoplasm (background). Bar: 10 µm.
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Grants and funding

This work has been supported by the Technology Agency of the Czech Republic (TA03010598 and TA03010719). The infrastructural part of the project (Institute of Molecular and Translational Medicine) has been supported from the Operational Program Research and Development for Innovations (project CZ.1.05/2.1.00/01.0030). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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