Conjugated polyelectrolyte amplified thiazole orange emission for label free sequence specific DNA detection with single nucleotide polymorphism selectivity.
We report a simple label-free method for sequence specific DNA detection using cationic conjugated polyelectrolyte (CCP) amplified thiazole orange (TO) emission as the signal. The discrimination of perfectly complementary DNA from a one-base mismatched sequence is accomplished by S1 nuclease digestion of the hybridized peptide nucleic acid (PNA)/DNA complexes. When the target DNA is complementary to the PNA probe, the DNA/PNA complexes remain after digestion, which allows TO intercalation to give fluorescence. Addition of CCP to this solution leads to enhanced TO emission due to fluorescence resonance energy transfer (FRET) from the CCP to TO, and the solution fluorescence appears yellow. In the presence of even one base mismatched DNA or double-stranded DNA molecules, S1 nuclease can effectively digest the DNA sequences into small fragments and no dye intercalation occurs. Addition of CCP to these solutions does not induce any polymer fluorescence change, and the solution fluorescent color is blue. This method allows visual detection of target DNA with a detection limit of 5 microM, which provides good groundwork for the future exploration of real-time instrument free single nucleotide polymorphism (SNP) diagnosis.
Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576, Singapore.
Kai
K
Li
Bin
B
Liu
D052160:Benzothiazoles / Q000737:chemistry*; D004573:Electrolytes / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015341:Nucleic Acid Probes / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011804:Quinolines / Q000737:chemistry*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D052160:Benzothiazoles; D004573:Electrolytes; D005456:Fluorescent Dyes; D015341:Nucleic Acid Probes; D011804:Quinolines; C051000:thiazole orange; D015719:Single-Strand Specific DNA and RNA Endonucleases