RESUMEN
An efficient strategy for nucleic acid labeling and analysis on deoxyribonucleic acid (DNA) chips has been developed. This approach, which combines the fragmentation and the labeling steps, is based on the reactivity of the phosphates of DNA and ribonucleic acid (RNA) fragments and is using reporter molecules bearing a bromomethyl- or aryldiazomethane-reactive group. In this chapter, we describe the preparation of the reactive label and protocols for efficient labeling of any nucleic acid sequence, DNA or RNA, prior to their hybridization, detection, and analysis on DNA chips.
Asunto(s)
Ácidos Nucleicos/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Hibridación de Ácido NucleicoRESUMEN
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.