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Magnetically assisted DNA assays: high selectivity using conjugated polymers for amplified fluorescent transduction.

Xu H, Wu H, Huang F, Song S, Li W, Cao Y, Fan C - Nucleic Acids Res. (2005)

Bottom Line: We demonstrate that the use of magnetic microparticles significantly improves the selectivity of this class of DNA sensors.Compared with previously reported DNA sensors with CP amplification, this novel sensing strategy displays excellent discrimination against non-cognate DNA in the presence of a protein mixture or even human serum.We also demonstrate that the magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

View Article: PubMed Central - PubMed

Affiliation: Division of Nanobiology and Nanomedicine, Shanghai Institute of Applied Physics, Chinese Academy of Sciences Shanghai 201800, China.

ABSTRACT
We report a strategy for conjugated polymer (CP)-based optical DNA detection with improved selectivity. The high sensitivity of CP-based biosensors arises from light harvesting by the CP and the related amplified fluorescent signal transduction. We demonstrate that the use of magnetic microparticles significantly improves the selectivity of this class of DNA sensors. Compared with previously reported DNA sensors with CP amplification, this novel sensing strategy displays excellent discrimination against non-cognate DNA in the presence of a protein mixture or even human serum. We also demonstrate that the magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

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Normalized fluorescence spectra for DNA hybridization with 1 nM perfectly matched (PM) target DNA, 1 nM single-nucleotide mismatched (SM) DNA and 5 μM non-cognate DNA of random sequence ([PF] = 8.1 × 10−8 M); 5 nM PM target DNA and 5 nM SM DNA ([PF] = 4.3 × 10−7 M).
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fig3: Normalized fluorescence spectra for DNA hybridization with 1 nM perfectly matched (PM) target DNA, 1 nM single-nucleotide mismatched (SM) DNA and 5 μM non-cognate DNA of random sequence ([PF] = 8.1 × 10−8 M); 5 nM PM target DNA and 5 nM SM DNA ([PF] = 4.3 × 10−7 M).

Mentions: We performed a stringent washing step that was expected to destabilize dsDNA containing single-nucleotide mismatches. In this case, MMPs collected from the test solution were rinsed with the washing buffer of low ionic strength. We reason that DNA sequences containing a single-nucleotide mismatch can be largely removed during this stringent washing, while target DNA remains bound to the MMP surfaces. Indeed, we observed that the DNA sensor showed marked differences in FRET signals corresponding to perfectly matched DNA and single-nucleotide mismatched DNA (Figure 3), indicating that this DNA sensor exhibits excellent discrimination against even a single-nucleotide mismatch.


Magnetically assisted DNA assays: high selectivity using conjugated polymers for amplified fluorescent transduction.

Xu H, Wu H, Huang F, Song S, Li W, Cao Y, Fan C - Nucleic Acids Res. (2005)

Normalized fluorescence spectra for DNA hybridization with 1 nM perfectly matched (PM) target DNA, 1 nM single-nucleotide mismatched (SM) DNA and 5 μM non-cognate DNA of random sequence ([PF] = 8.1 × 10−8 M); 5 nM PM target DNA and 5 nM SM DNA ([PF] = 4.3 × 10−7 M).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1131937&req=5

fig3: Normalized fluorescence spectra for DNA hybridization with 1 nM perfectly matched (PM) target DNA, 1 nM single-nucleotide mismatched (SM) DNA and 5 μM non-cognate DNA of random sequence ([PF] = 8.1 × 10−8 M); 5 nM PM target DNA and 5 nM SM DNA ([PF] = 4.3 × 10−7 M).
Mentions: We performed a stringent washing step that was expected to destabilize dsDNA containing single-nucleotide mismatches. In this case, MMPs collected from the test solution were rinsed with the washing buffer of low ionic strength. We reason that DNA sequences containing a single-nucleotide mismatch can be largely removed during this stringent washing, while target DNA remains bound to the MMP surfaces. Indeed, we observed that the DNA sensor showed marked differences in FRET signals corresponding to perfectly matched DNA and single-nucleotide mismatched DNA (Figure 3), indicating that this DNA sensor exhibits excellent discrimination against even a single-nucleotide mismatch.

Bottom Line: We demonstrate that the use of magnetic microparticles significantly improves the selectivity of this class of DNA sensors.Compared with previously reported DNA sensors with CP amplification, this novel sensing strategy displays excellent discrimination against non-cognate DNA in the presence of a protein mixture or even human serum.We also demonstrate that the magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

View Article: PubMed Central - PubMed

Affiliation: Division of Nanobiology and Nanomedicine, Shanghai Institute of Applied Physics, Chinese Academy of Sciences Shanghai 201800, China.

ABSTRACT
We report a strategy for conjugated polymer (CP)-based optical DNA detection with improved selectivity. The high sensitivity of CP-based biosensors arises from light harvesting by the CP and the related amplified fluorescent signal transduction. We demonstrate that the use of magnetic microparticles significantly improves the selectivity of this class of DNA sensors. Compared with previously reported DNA sensors with CP amplification, this novel sensing strategy displays excellent discrimination against non-cognate DNA in the presence of a protein mixture or even human serum. We also demonstrate that the magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

Show MeSH