Duplexes, spotting

This assay can also be used to determine intra- and inter-array variabilities (see Note 10). p53 is a tumor suppressor protein and, as part of its function as a transcription factor, it has a specific DNA-binding domain (21,22). The DNA-binding assay therefore comprises binding a Cy3-labeled duplex DNA containing a p53 recognition site to the active immobilized p53 proteins, and provides a measurement of activity of the proteins immobilized in each spot. 

Fig. 6. Measurement of the relative amount of ligand bound to each protein in the array. (A) Schematic of on-chip binding assay in which a fluorescently labeled interaction partner binds to the functional, arrayed protein immobilized to the streptavidin-coated surface via the biotinylated BCCP tag. (B) p53 protein function microarray probed with Cy3-labeled GADD45 duplex oligo. Quantification of the signal intensity from each spot allows the effect of polymorphic and functional variation on the DNA binding function of p53 to be determined.

Benzo[a]pyrene diol epoxides react with adenine residues in DNA to give 105(-l-)- and 10R(-)-tra s-anti-benzo[a]pyrene-N -dA adducts (103), lOR(-) shown, which give rise to mutational hot spots. The solution structure of an 11-mer duplex in which there is a modified adenine has been studied, and it was... 

A typical experiment then consists of preparing the surface and placing the probes on the surface (often not in the experimenter s lab) followed by placing the sample saline solution containing the target on the spots. The kinetics of hybridization may then proceed. Once sufficient time has elapsed, saline solution is used to wash the remaining unhybridized nucleic acid away. At this point the fluorophores can be excited and the area normalized intensity of fluorescence at each spot is then related to the affinity and the concentration of duplex nucleic acid. 

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