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.

• MeSH
• buněčné jádro genetika   MeSH
• DNA-polymerasa I metabolismus   MeSH
• DNA genetika   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mitochondriální DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA-polymerasa I MeSH
• DNA MeSH
• mitochondriální DNA MeSH

A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-2'-deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off.

• MeSH
• barvení a značení MeSH
• bromodeoxyuridin chemie   metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• deoxyribonukleasa I MeSH
• fluorescenční protilátková technika nepřímá MeSH
• HeLa buňky MeSH
• kyselina askorbová chemie   MeSH
• kyslík chemie   MeSH
• lidé MeSH
• mitochondriální DNA chemie   genetika   MeSH
• oxidace-redukce MeSH
• replikace DNA * MeSH
• síran měďnatý chemie   MeSH
• štěpení DNA * MeSH
• superoxiddismutasa chemie   MeSH
• superoxidy chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bromodeoxyuridin MeSH
• deoxyribonukleasa I MeSH
• kyselina askorbová MeSH
• kyslík MeSH
• mitochondriální DNA MeSH
• síran měďnatý MeSH
• superoxiddismutasa MeSH
• superoxidy MeSH

5-Bromo-2'-deoxyuridine (BrdU) and 2'-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.

• MeSH
• afinita protilátek MeSH
• barvení a značení MeSH
• biologický transport MeSH
• biotinylace MeSH
• bromodeoxyuridin chemie   imunologie   metabolismus   MeSH
• deoxyuridin analogy a deriváty   chemie   imunologie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• fluorescenční barviva MeSH
• fluorescenční mikroskopie * MeSH
• HeLa buňky MeSH
• lidé MeSH
• protilátky chemie   imunologie   MeSH
• zkřížené reakce imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5-ethynyl-2'-deoxyuridine MeSH Prohlížeč
• bromodeoxyuridin MeSH
• deoxyuridin MeSH
• DNA MeSH
• fluorescenční barviva MeSH
• protilátky MeSH