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New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

Gbaj A, Bichenkova E, Walsh L, Savage H, Sardarian A, Etchells L, Gulati A, Hawisa S, Douglas K - Libyan J Med (2009)

Bottom Line: The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis.Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies.Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

View Article: PubMed Central - PubMed

Affiliation: Wolfson Centre for Rational Structure-Based Design of Molecular Diagnostics, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, UK.

ABSTRACT
The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5'-bispyrene and 3'-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5'-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

No MeSH data available.


Related in: MedlinePlus

Fluorescence spectra of 5′-bispyrene alone (1), 5′- bispyrene and target (2), and 5′-bispyren, target and 3′- naphthalene (3) in 0% TFE/ Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. Component concentrations were 2.5 µM in a total volume of 100 µL. Excitation was at 350 nm, spectra are corrected for TFE and buffer.
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Figure 0004: Fluorescence spectra of 5′-bispyrene alone (1), 5′- bispyrene and target (2), and 5′-bispyren, target and 3′- naphthalene (3) in 0% TFE/ Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. Component concentrations were 2.5 µM in a total volume of 100 µL. Excitation was at 350 nm, spectra are corrected for TFE and buffer.

Mentions: Figure 4 shows the broad structure-less peak at 480 nm and also smaller distinct peaks at 379 nm and 397 nm, which are due to the monomer that was not previously identified in 80% TFE. The intensity of excimer emission (at 480 nm) is much lower at 0% than that at 80% TFE, and the monomer peaks are much more obvious. These advantages make the aqueous medium preferable because identification of target depends on changes of both monomer and excimer peaks not just the excimer peak.


New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

Gbaj A, Bichenkova E, Walsh L, Savage H, Sardarian A, Etchells L, Gulati A, Hawisa S, Douglas K - Libyan J Med (2009)

Fluorescence spectra of 5′-bispyrene alone (1), 5′- bispyrene and target (2), and 5′-bispyren, target and 3′- naphthalene (3) in 0% TFE/ Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. Component concentrations were 2.5 µM in a total volume of 100 µL. Excitation was at 350 nm, spectra are corrected for TFE and buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3066750&req=5

Figure 0004: Fluorescence spectra of 5′-bispyrene alone (1), 5′- bispyrene and target (2), and 5′-bispyren, target and 3′- naphthalene (3) in 0% TFE/ Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. Component concentrations were 2.5 µM in a total volume of 100 µL. Excitation was at 350 nm, spectra are corrected for TFE and buffer.
Mentions: Figure 4 shows the broad structure-less peak at 480 nm and also smaller distinct peaks at 379 nm and 397 nm, which are due to the monomer that was not previously identified in 80% TFE. The intensity of excimer emission (at 480 nm) is much lower at 0% than that at 80% TFE, and the monomer peaks are much more obvious. These advantages make the aqueous medium preferable because identification of target depends on changes of both monomer and excimer peaks not just the excimer peak.

Bottom Line: The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis.Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies.Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

View Article: PubMed Central - PubMed

Affiliation: Wolfson Centre for Rational Structure-Based Design of Molecular Diagnostics, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, UK.

ABSTRACT
The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5'-bispyrene and 3'-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5'-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

No MeSH data available.


Related in: MedlinePlus