Base pairing, electrochemical detection, nucleic

Importantly, King succeeded in the formation of a Fc-labeled 998-bp (base-pair) construct by PCR using T4 DNA polymerase in the presence of Fcl-dUTP. Incorporation of the redox label shows that Fcl-dUTP is suitable as a substrate for PCR. In contrast, Fc2-dUTP acts predominantly as a terminator in the PCR. The melting behavior of a 37-mer duplex containing five Fcl-dU residues reveals that the labeled nucleotide induces only a modest helix destabilisation, with T ,=71°C for a labeled duplex versus 75°C for the corresponding nonlabeled ds-DNA construct. King reports that the Fc-labeled DNA is detected at femtomolar levels by HPLC using a coulometric detector. Thus, it must be emphasized that the incorporation of the Fc label by PCR and its facile and cost-effective electrochemical detection should promote the use of this technique in nucleic acid analysis and may replace the more costly fluorescence-based detection systems. 

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