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.
Highlights
Many approaches are used for the visualization of DNA molecules in situ
We have previously shown that the incubation of formaldehydefixed and Triton X100-permeabilized cells with copper(I) ions leads to the formation of frequent gaps in DNA [8]
The main advantage of the approach presented is the absolute selectivity of the double-stranded DNA, the insensitivity to the presence of RNA and the possibility to reveal the mitochondrial genome
Summary
Many approaches are used for the visualization of DNA molecules in situ In this respect, substances that bind to DNA and concurrently serve as fluorescent markers are most frequently used for the labeling of double-stranded DNA. These include substances that bind to the minor groove of DNA, for example DAPI or Hoechst dyes [1,2], or substances that intercalate in the double-stranded DNA, such as YOYO-1 or TOTO-1 [3,4]. They include a relatively time-consuming approach based on in-situ hybridization [6], immunocytochemical detection by anti-dsDNA antibodies [7] or detection of incorporated 5-bromo-29-deoxyuridine (BrdU) by anti-BrdU antibodies [8,9]. Unlike BrdU, EdU obligatorily requires an amplification step [11,12,13]
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