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The San Diego Conference Nucleic Acid Technologies in Disease Detection November 17–19, 1999

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Direct Electrochemical Detection of Nucleic Acids. Jill C. Mikulecky, Robert S. Thomas, Allen E. Eckhardt, Mary E. Napier, Natasha D. Popovich, Jonathan L. Baron, Mark D. Gelardi, XANTHON, Inc., Research Triangle Park, NC 27709.

The detection of target nucleic acid hybridized to complementary capture probe is of central importance in genomic research and pathogen diagnostics. Present detection methods require attachment of enzymatic, fluorescent, chemiluminescent, or radioactive labels to the hybridized target. Furthermore, these methods require amplification of rare targets to detect low copies of a specific nucleic acid. These procedures are costly and time consuming, require complex instrumentation, and may introduce sample contamination. We have developed a novel detection method for DNA and RNA that does not require reporter molecule attachment or target amplification. The target nucleic acid is electrochemically detected using a soluble, metal mediator, tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)₃²⁺). When the appropriate potential is applied, Ru(bpy)₃²⁺ oxidizes guanine via a catalytic cycle and current is produced. This method, unlike other electrochemical techniques, does not require the target to be directly wired to the electrode or the use of duplex specific redox-active indicators.

The present detector format utilizes a 6 mm diameter electrode, upon which a monolayer pre-coupled with oligonucleotide probe is self assembled. Complementary nucleic acid target is allowed to hybridize to the probe, unhybridized target washed away, Ru(bpy)₃²⁺ added, and the signal acquired. Using this format, 100 femtomoles of a synthetic 124-mer oligonucleotide target containing 41 guanines generated a current of 0.18 mA over background. To improve sensitivity, the detector format is transitioning to a 200 micron diameter electrode, where approximately 100 attomoles of a synthetic 21-mer oligonucleotide containing 5 guanines generated a current of 600 nA over background.

The specificity and efficiency of the system is being optimized using synthetic oligonucleotides, PCR products, . . . [Full Text of this Article]

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