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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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Related in: MedlinePlus

Specificity of oligodeoxyribonucleotide hybridization to PNA probes. Hybridizations were performed at room temperature with a concentration of 7.5 × 1010 targets per μL using the fluorescent cationic polymer for detection. Hybridization of PNA probes to perfectly complementary, or complementary oligonucleotides presenting a terminal mismatch, a central mismatch, or two mismatches were performed in triplicate. Fluorescence intensities from hybridized probes were corrected by substraction of background fluorescence intensity.
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Figure 2: Specificity of oligodeoxyribonucleotide hybridization to PNA probes. Hybridizations were performed at room temperature with a concentration of 7.5 × 1010 targets per μL using the fluorescent cationic polymer for detection. Hybridization of PNA probes to perfectly complementary, or complementary oligonucleotides presenting a terminal mismatch, a central mismatch, or two mismatches were performed in triplicate. Fluorescence intensities from hybridized probes were corrected by substraction of background fluorescence intensity.

Mentions: Specificity of detection was investigated by hybridizing mismatched oligonucleotides to PNA probes. After room temperature hybridization of oligonucleotides with PNA probes, the fluorescent polythiophene polymeric biosensor gave a strong signal over background when target oligonucleotide was fully complementary to the capture probe. Oligonucleotides with two mismatches and non complementary oligonucleotides produced near-background signals easily distinguishable from the much stronger signal observed with perfectly matched hybrids (21-fold stronger) (Figure 2). For a single mismatch, discrimination is related to the position of the mismatch in the capture PNA probe. When the mismatch is located at the probe extremity, signal intensity is reduced 2.5 fold as compared to the perfect match. By contrast, a ratio of 6 is observed when the mismatch is located close to the center of the capture probe (Figure 2).


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)

Specificity of oligodeoxyribonucleotide hybridization to PNA probes. Hybridizations were performed at room temperature with a concentration of 7.5 × 1010 targets per μL using the fluorescent cationic polymer for detection. Hybridization of PNA probes to perfectly complementary, or complementary oligonucleotides presenting a terminal mismatch, a central mismatch, or two mismatches were performed in triplicate. Fluorescence intensities from hybridized probes were corrected by substraction of background fluorescence intensity.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Specificity of oligodeoxyribonucleotide hybridization to PNA probes. Hybridizations were performed at room temperature with a concentration of 7.5 × 1010 targets per μL using the fluorescent cationic polymer for detection. Hybridization of PNA probes to perfectly complementary, or complementary oligonucleotides presenting a terminal mismatch, a central mismatch, or two mismatches were performed in triplicate. Fluorescence intensities from hybridized probes were corrected by substraction of background fluorescence intensity.
Mentions: Specificity of detection was investigated by hybridizing mismatched oligonucleotides to PNA probes. After room temperature hybridization of oligonucleotides with PNA probes, the fluorescent polythiophene polymeric biosensor gave a strong signal over background when target oligonucleotide was fully complementary to the capture probe. Oligonucleotides with two mismatches and non complementary oligonucleotides produced near-background signals easily distinguishable from the much stronger signal observed with perfectly matched hybrids (21-fold stronger) (Figure 2). For a single mismatch, discrimination is related to the position of the mismatch in the capture PNA probe. When the mismatch is located at the probe extremity, signal intensity is reduced 2.5 fold as compared to the perfect match. By contrast, a ratio of 6 is observed when the mismatch is located close to the center of the capture probe (Figure 2).

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