PCR-independent Reagent-Free Nucleic Acid Detection

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DESCRIPTION (provided by applicant): The overall goal of this project is to demonstrate the feasibility of 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 100 nm-diameter beads. 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 ~100 nm-diameter polystyrene 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 across a solid-state membrane harboring a nanopore. However, the substantial negative charge acquired upon capture of a target DNA or RNA sequence would make the hybridized conjugate electrophoretically mobile. If the nanopore size tapers to a diameter smaller than 100 nm, the charged conjugate carrying the hybridized PNA and target NA would be expected to enter the large end of the pore 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. 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. PUBLIC HEALTH RELEVANCE: We propose to develop a device for the detection of specific nucleic acid sequences based on the electrical measurement of blockage of a nanopore by a bead to which the nucleic acid has bound. The device produces a strong, essentially binary, signal that can be easily reported with simple circuitry, an approach with major advantages over conventional PCR and fluorescent-based nucleic acid detection technologies. Potential applications of this technology include: infectious disease diagnostics, genetic screening for hereditary diseases, personalized medicine, criminology, paternity disputes, animal and plant breeding, patient screening during epidemics, oncological status assessment during surgery, detection of food contaminants, and biowarfare agent detection.