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22 February 2008 Highly specific identification of single nucleic polymorphism in M. tuberculosis using smart probes and single-molecule fluorescence spectroscopy in combination with blocking oligonucleotides
Achim Friedrich, Matthias Müller, Oliver Nolte, Jürgen Wolfrum, Markus Sauer, Jörg D. Hoheisel, Jens-Peter Knemeyer, Nicole Marme
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Proceedings Volume 6867, Molecular Probes for Biomedical Applications II; 68670N (2008) https://doi.org/10.1117/12.763077
Event: SPIE BiOS, 2008, San Jose, California, United States
Abstract
In this article we present a method for the highly specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis. This approach applies fluorescently labeled hairpin-structured oligonucleotides (smart probes) and confocal single-molecule fluorescence spectroscopy. Smart probes are fluorescently labeled at the 5'-end. The dye's fluorescence is quenched in the closed hairpin conformation due to close proximity of the guanosine residues located at the 3'-end. As a result of the hybridization to the complementary target sequence the hairpin structure and thus fluorescence quenching gets lost and a strong fluorescence increase appears. To enhance the specificity of the SNP detection unlabeled "blocking oligonucleotides" were added to the sample. These oligonucleotides hybridizes to the DNA sequence containing the mismatch thus masking this sequence and hereby preventing the smart probe from hybridizing to the mismatched sequence.
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Achim Friedrich, Matthias Müller, Oliver Nolte, Jürgen Wolfrum, Markus Sauer, Jörg D. Hoheisel, Jens-Peter Knemeyer, and Nicole Marme "Highly specific identification of single nucleic polymorphism in M. tuberculosis using smart probes and single-molecule fluorescence spectroscopy in combination with blocking oligonucleotides", Proc. SPIE 6867, Molecular Probes for Biomedical Applications II, 68670N (22 February 2008); https://doi.org/10.1117/12.763077
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KEYWORDS
Luminescence
Fluorescence spectroscopy
Resistance
Molecules
Simulation of CCA and DLA aggregates
Confocal microscopy
Diagnostics
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