Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 1, Pages 34-39Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0407578101
Keywords
biosensor; energy transfer; hybridization probes; polyelectrolyte; fluorescence
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Funding
- NIGMS NIH HHS [R01 GM062958, GM 62958-01] Funding Source: Medline
- NINDS NIH HHS [NS 35010, R01 NS035010] Funding Source: Medline
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A strategy employing a combination of peptide nucleic acid (PNA) probes, an optically amplifying conjugated polymer (CP), and S1 nuclease enzyme is capable of detecting SNPs in a simple, rapid, and sensitive manner. The recognition is accomplished by sequence-specific hybridization between the uncharged, fluorescein-labeled PNA probe and the DNA sequence of interest. After subsequent treatment with S1 nuclease, the cationic water soluble CP electrostatically associates with the remaining anionic PNA/DNA complex, leading to sensitized emission of the labeled PNA probe via FRET from the CP. The generation of fluorescent signal is controlled by strand-specific electrostatic interactions and is governed by the complementarity of the probe/target pair. To assess the method, we compared the ability of the sensor system to detect normal, wild-type human DNA sequences, and those sequences containing a single base mutation. Specifically, we examined a PNA probe complementary to a region of the gene encoding the microtubule associated protein tau. The probe sequence covers a known point mutation implicated in a dominant neurodegenerative dementia known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), which has clinical and molecular similarities to Alzheimer's disease. By using an appropriate PNA probe, the conjugated polymer poly[(9,9-bis(6'-N,N,N-trimethylammoniumhexylbromide)fluorene)-co-phenylene] and S1 nuclease, unambiguous FRET signaling is achieved for the wildtype DNA and not the mutant sequence harboring the SNP. Distance relationships in the CP/PNA assay are also discussed to highlight constraints and demonstrate improvements within the system.
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