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Proceedings Paper

Highly specific identification of single nucleic polymorphism in M. tuberculosis using smart probes and single-molecule fluorescence spectroscopy in combination with blocking oligonucleotides
Author(s): Achim Friedrich; Matthias Müller; Oliver Nolte; Jürgen Wolfrum; Markus Sauer; Jörg D. Hoheisel; Jens-Peter Knemeyer; Nicole Marme
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Paper 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.

Paper Details

Date Published: 22 February 2008
PDF: 8 pages
Proc. SPIE 6867, Molecular Probes for Biomedical Applications II, 68670N (22 February 2008); doi: 10.1117/12.763077
Show Author Affiliations
Achim Friedrich, German Cancer Research Ctr. (Germany)
Matthias Müller, Univ. of Heidelberg (Germany)
Oliver Nolte, Univ. of Heidelberg (Germany)
Jürgen Wolfrum, Univ. of Heidelberg (Germany)
Markus Sauer, Univ. of Bielefeld (Germany)
Jörg D. Hoheisel, German Cancer Research Ctr. (Germany)
Jens-Peter Knemeyer, German Cancer Research Ctr. (Germany)
Nicole Marme, Univ. of Heidelberg (Germany)

Published in SPIE Proceedings Vol. 6867:
Molecular Probes for Biomedical Applications II
Samuel Achilefu; Darryl J. Bornhop; Ramesh Raghavachari, Editor(s)

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