SNP detection using peptide nucleic acid probes and conjugated polymers: applications in neurodegenerative disease identification.
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 wild-type 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.
Gaylord
Brent S
BS
Materials Department and Institute for Polymers and Organic Solids and Neuroscience Research Institute, University of California-Santa Barbara, Santa Barbara, CA 93106, USA.
Massie
Michelle R
MR
Feinstein
Stuart C
SC
Bazan
Guillermo C
GC
D003627:Data Interpretation, Statistical; D006801:Humans; D015335:Molecular Probes; D019636:Neurodegenerative Diseases / Q000175:diagnosis*; D020135:Peptide Nucleic Acids; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence; D016875:tau Proteins / Q000235:genetics
D015335:Molecular Probes; D020135:Peptide Nucleic Acids; D016875:tau Proteins