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Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support.

Raymond FR, Ho HA, Peytavi R, Bissonnette L, Boissinot M, Picard FJ, Leclerc M, Bergeron MG - BMC Biotechnol. (2005)

Bottom Line: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray.This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries.This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Recherche en Infectiologie, l'Université Laval, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, Sainte-Foy, Québec, G1V 4G2, Canada. fraymond@scientist.com

ABSTRACT

Background: Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization.

Results: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray.

Conclusion: This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

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Cationic polythiophene transducer for the fluorometric detection of hybridization on microarrays. A) Schematic depiction of the interaction between cationic polymers and a) single-stranded DNA, b) double-stranded DNA, c) single-stranded PNA and d) PNA-DNA duplex. Fluorescent cationic polymer is shown in yellow, DNA probes are shown in green and PNA probes are shown in red. B) Experimental results for fluorometric detection on microarray when cationic polythiophene transducer is reacted with a) single-stranded DNA, b) double-stranded DNA, c) single-stranded PNA and d) PNA-DNA duplex. Results are shown in triplicate. C) Graphs showing the fluorescence intensity with standard deviation for each triplicate shown in B.
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Figure 1: Cationic polythiophene transducer for the fluorometric detection of hybridization on microarrays. A) Schematic depiction of the interaction between cationic polymers and a) single-stranded DNA, b) double-stranded DNA, c) single-stranded PNA and d) PNA-DNA duplex. Fluorescent cationic polymer is shown in yellow, DNA probes are shown in green and PNA probes are shown in red. B) Experimental results for fluorometric detection on microarray when cationic polythiophene transducer is reacted with a) single-stranded DNA, b) double-stranded DNA, c) single-stranded PNA and d) PNA-DNA duplex. Results are shown in triplicate. C) Graphs showing the fluorescence intensity with standard deviation for each triplicate shown in B.

Mentions: Fluorescent conjugated polythiophene derivative used in this study binds nucleic acids via electrostatic interactions with negatively-charged phosphate groups of the DNA backbone [4,5]. This is illustrated in Figure 1 where single-stranded and double-stranded DNA oligonucleotides (Figure 1a and 1b) both produce fluorescent signals due to the formation of DNA-polythiophene complexes. Thus, discrimination between hybridized and non-hybridized DNA capture probes is not possible using a conventional microarray fluorescence scanner.


Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support.

Raymond FR, Ho HA, Peytavi R, Bissonnette L, Boissinot M, Picard FJ, Leclerc M, Bergeron MG - BMC Biotechnol. (2005)

Cationic polythiophene transducer for the fluorometric detection of hybridization on microarrays. A) Schematic depiction of the interaction between cationic polymers and a) single-stranded DNA, b) double-stranded DNA, c) single-stranded PNA and d) PNA-DNA duplex. Fluorescent cationic polymer is shown in yellow, DNA probes are shown in green and PNA probes are shown in red. B) Experimental results for fluorometric detection on microarray when cationic polythiophene transducer is reacted with a) single-stranded DNA, b) double-stranded DNA, c) single-stranded PNA and d) PNA-DNA duplex. Results are shown in triplicate. C) Graphs showing the fluorescence intensity with standard deviation for each triplicate shown in B.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1131893&req=5

Figure 1: Cationic polythiophene transducer for the fluorometric detection of hybridization on microarrays. A) Schematic depiction of the interaction between cationic polymers and a) single-stranded DNA, b) double-stranded DNA, c) single-stranded PNA and d) PNA-DNA duplex. Fluorescent cationic polymer is shown in yellow, DNA probes are shown in green and PNA probes are shown in red. B) Experimental results for fluorometric detection on microarray when cationic polythiophene transducer is reacted with a) single-stranded DNA, b) double-stranded DNA, c) single-stranded PNA and d) PNA-DNA duplex. Results are shown in triplicate. C) Graphs showing the fluorescence intensity with standard deviation for each triplicate shown in B.
Mentions: Fluorescent conjugated polythiophene derivative used in this study binds nucleic acids via electrostatic interactions with negatively-charged phosphate groups of the DNA backbone [4,5]. This is illustrated in Figure 1 where single-stranded and double-stranded DNA oligonucleotides (Figure 1a and 1b) both produce fluorescent signals due to the formation of DNA-polythiophene complexes. Thus, discrimination between hybridized and non-hybridized DNA capture probes is not possible using a conventional microarray fluorescence scanner.

Bottom Line: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray.This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries.This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Recherche en Infectiologie, l'Université Laval, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, Sainte-Foy, Québec, G1V 4G2, Canada. fraymond@scientist.com

ABSTRACT

Background: Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization.

Results: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray.

Conclusion: This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

Show MeSH
Related in: MedlinePlus