Enzymatic labels

Regardless of the type of enzymatic labeling used, it is important that the label be incorporated into the nucleoside triphosphates or primers in a way that does not affect enzyme recognition and activity. Thus, every enzymatic labeling procedure for modifying RNA or DNA probes must start with chemical derivatization of individual nucleotides. Of the many chemical procedures that can be used to modify a nucleoside triphosphate monomer, there are only a few that will result in a derivative still able to be enzymatically added to an existing oligonucleotide strand. 

The pyrimidine nucleosides dUTP or dCTP can be modified at their C-5 position with a spacer arm containing a tag, such as a biotin group, and still remain good substrates for DNA polymerase. Enzymatic labeling with a biotin-modified pyrimidine nucleoside triphosphate is one of the most common methods of adding a detectable group to an existing DNA strand. 

However, luminescence-based detection techniques often require a high number of steps. Consider ELISA as an example. As a first step, the sample is introduced into a 96-well plate an antibody targeting the antigen of interest has been immobilized to the wells of the plate. After a rinse, the wells contain the antibody and any bound antigen. However, although the antigen has been isolated, the protocol is nowhere near completion. The remaining steps include another antibody (different from the first) to form a sandwich assay, a secondary antibody with an enzymatic label, and a substrate that is luminescent when activated by the enzyme. Finally, the sample is analyzed by relatively expensive detection optics to determine the amount of analyte that was captured in the assay. The steps are illustrated in Fig. 14.1a. 

Saiki, R., Bugawan, T.L., Horn, G.T., Mullis, K.B., Ehrlich, H.A. (1986) The analysis of enzymatically labeled / -globin DQa DNA with allele-specific oligonucleotide probes. Nature 324, 163-166. 

Enzymatic labeling using any of these polymerase methods results in derivatized nucleoside triphosphates being incorporated at numerous locations within an oli-... 

Once immobilized on GEC, DNA preserves its unique hybridization properties, which can be revealed using different strategies based on both enzymatic labelling and the intrinsic signal coming from the DNA oxidation. 

Fig. 21.4. Schematic representation for the detection of Salmonella through (1A) a rapid verification of PCR amplification based on the doubly labelled PCR product detection and (IB) real-time PCR reactor based on PCR amplification with magnetic bead primers on streptavidin-modified magnetic beads. (2) Enzymatic labelling (3) magnetic capture of the modified magnetic beads by the magneto electrode (m-GEC) and (4) chronoamperometric determination are common steps for all of these strategies (1A, IB).

The hybridization event can be detected both with label-free or enzymatic labelling procedures. The single-point attachment of DNA can be achieved by the immobilization of biotinylated DNA on Av-GEB platform. In this case, a one-step immobilization/hybridization procedure is achieved. The capability of surface regeneration of the biocomposite electrodes allows repeated analyses with the same electrode as... 

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