Electronic detection of single-base mismatches in DNA with ferrocene-modified probes

Genotyping and gene-expression monitoring is critical to the study of the a ssociation between genetics and drug response (pharmacogenomics) and the as sociation of sequence variation with heritable phenotypes. Recently, we dev eloped an entirely electronic method for the detection of DNA hybridization events by the site-specific incorporation of ferrocenyl derivatives into D NA oligonucleotides. To pet-form rapid and accurate point mutation detectio n employing this methodology, two types of metal-containing signaling probe s with varying redox potentials are required. In this report we describe a new ferrocene-containing phosphoramidite 9 that provides a range of detecta ble redox potentials. Using automated DNA/RNA synthesis techniques the two ferrocenyl complexes were inserted at various positions along oligonucleoti de probes. Thermal stability analysis of these metal-containing DNA oligonu cleotides indicates that incorporation of 9 resulted in no destabilization of the duplex. A mixture of oligonucleotides containing compounds 9 and I w as analyzed by alternating current voltammetry (ACV) monitored at the Ist h armonic. The data demonstrate that the two ferrocenyl oligonucleotide deriv atives can be distinguished electrochemically. A CMS-DNA array was prepared on an array of gold electrodes on a printed circuit board substrate with a self-assembled mixed monolayer, coupled to an electronic detection system. Experiments for the detection of a single-base match utilizing two signali ng probes were carried out. The results demonstrate that rapid and accurate detection of a single-base mismatch can be achieved by using these dual-si gnaling probes on CMS-DNA chips.