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Electrochemical analysis of DNA using switchable redox moieties
Fojta, Miroslav ; Daňhel, Aleš ; Horáková Brázdilová, Petra ; Plucnara, Medard ; Pivoňková, Hana ; Havran, Luděk ; Vidláková, Pavlína ; Raindlová, Veronika ; Balintová, Jana ; Macíčková-Cahová, Hana ; Hocek, Michal
Labelling of DNA with electrochemically active moieties proved to be a convenient way to the development of electrochemical techniques for the sequence-specific DNA sensing. Through combinations of various labels differing in redox potentials, independent redox coding of different DNA sequences or individual nucleobases can be attained. Applications possibilities of electrochemistry in analysis of modified DNAs are further extended by facile monitoring of chemical conversion of reactive groups on DNA during post-labelling with ultimate redox labels. In addition, controlled in situ electrochemical conversions of specific intrinsic and extrinsic DNA components can be utilized to switch their electrochemical signals and improve signal resolution.
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Polymerase synthesis of base-modified DNA: New methods and new applications
Balintová, Jana ; Daďová, Jitka ; Kielkowski, Pavel ; Ménová, Petra ; Vaníková, Zuzana ; Riedl, Jan ; Raindlová, Veronika ; Fojta, Miroslav ; Hocek, Michal
Diverse base-modified oligonucleotides and double-stranded DNA molecules were prepared by polymerase incorporation of modified nucleoside triphosphates. The methods include primer extension, PCR, nicking enzyme amplification reaction and mixed incorporations. The modified nucleic acids were used in redox and fluorescence labelling and coding, as well as regulation of binding and cross-linking with proteins.
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Redox labelling of nucleic acids for analyzing nucleotide sequences and monitoring DNA-protein interactions
Fojta, Miroslav ; Havran, Luděk ; Horáková Brázdilová, Petra ; Pivoňková, Hana ; Kostečka, Pavel ; Macíčková-Cahová, Hana ; Raindlová, Veronika ; Vrábel, Milan ; Hocek, Michal
Nucleobase labelling of DNA for electrochemical sensing was attained through chemical modification of thymine bases with osmium tetroxide in the presence of nitrogenous ligands, or via enzymatic incorporation of nucleotide conjugates with redox-active moieties using labelled deoxynucleoside triphosphates. DNA hybridization, primer extension and PCR techniques were used for sequence-specific DNA assays. Tail-labelled DNA substrates were applied to monitor DNA binding by tumour suppressor p53 protein.
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Direct enzymatic synthesis of aldehyde-functionalized DNA and its conjugation with hydrazines and amines
Raindlová, Veronika ; Hocek, Michal
A new simple methodology for DNA conjugation or staining was developed. 2′-Deoxyribonucleoside triphosphates (dNTPs) bearing reactive aldehyde group were prepared by one-step Suzuki cross-coupling reaction of halogenated dNTPs with boronic acid. These modified dNTPs were enzymatically incorporated into DNA by primer extension (PEX) or amplified by polymerase chain reaction (PCR) using different DNA polymerases. The followup reaction between aldehyde-modified PCR products and hydrazine derivatives gave coloured DNA conjugated hydrazones. This methodology was also used for further conjugations of aldehyde-modified 2′-deoxyribonucleoside monophosphates (dNMPs) with amines by reductive amination.
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