(680c) PCR-Independent, Reagent-Free, Binary-Mode Nucleic Acid Detection

A new concept for specific nucleic acid (NA) sequence detection that does not rely on polymerase chain reaction (PCR) for target sequence amplification and does not require any special reagents other than a complementary sequence capture probe conjugated to polymer spheres has been demonstrated. Recent work has shown that the presence of single macromolecules in a nanopore causes measurable changes in the pore’s electrical resistance. Our concept takes this single-molecule detection system a significant step further by amplifying the signals from specific single molecule targets to generate an easily detected on/off current signal due to a very large sustained increase in pore resistance. The key elements of the proposed system include a peptide nucleic acid (PNA) capture probe conjugated to polymer spheres. PNA oligomers are uncharged analogs to DNA and RNA that share the same base chemistry and hybridize strongly to complementary NA sequences. Since the sphere-PNA conjugates carry little or no charge, they do not exhibit electrophoretic movement in response to a steady, DC field imposed through a pore. However, the substantial negative charge acquired upon capture of a target DNA or RNA sequence makes the hybridized conjugate electrophoretically mobile. If the pore size tapers to a diameter smaller than the spheres, the charged conjugate carrying the hybridized PNA and target NA would be expected to enter the large end of the pore, block it, and significantly increase its resistance, thereby causing a very strong, sustained drop in measured current. In such a way, this system is expected to give an essentially binary response signaling the absence or presence of a target NA. To date, the concept has been demonstrated at the microscale. In so doing, we have successfully detected ssDNA with a 12-mer portion of a gene encoding the anthrax lethal factor. This proposed new technology would be useful for applications where determination of the presence or absence of NA of a particular sequence, rather than its concentration, is of primary concern such as in patient screening during epidemics, oncological status assessment during surgery, detection of food contaminants, and biowarfare agent detection.