RESUMO
Molecules 2-5 that include in their structure a biotin moiety as detectable unit and differently substituted phenyl diazo functions as reactive group were prepared as reagents for labeling the phosphate group in mono and polynucleotides. These molecules were shown to react selectively and quantitatively with the model nucleotide 3'-UMP. They were used successfully in the labeling step of DNA and RNA analysis using high-density DNA-chips (or microarrays) technology.
Assuntos
Biotina/química , Diazometano/química , Nucleotídeos/química , Fosfatos/química , Eletroforese Capilar , Hidrólise , Espectroscopia de Ressonância MagnéticaRESUMO
DNA and RNA labeling and detection are key steps in nucleic acid-based technologies, used in medical research and molecular diagnostics. We report here the synthesis, reactivity, and potential of a new type of labeling molecule, m-(N-Biotinoylamino)phenylmethyldiazomethane (m-BioPMDAM), that reacts selectively and efficiently with phosphates in nucleotide monomers, oligonucleotides, DNA, and RNA. This molecule contains a biotin as detectable unit and a diazomethyl function as reactive moiety. We demonstrate that this label fulfills the requirements of stability, solubility, reactivity, and selectivity for hybridization-based analysis and especially for detection on high-density DNA chips.
Assuntos
DNA/análise , Diazometano/análogos & derivados , Ácidos Nucleicos/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biotinilação , DNA/química , Diazometano/síntese química , Corantes Fluorescentes , Indicadores e Reagentes , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Hibridização de Ácido Nucleico , RNA/análise , RNA/químicaRESUMO
An efficient and direct labeling method based on direct alkylation of nucleic acids at phosphates by aryldiazomethane derivatives is described.
