Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

Marme N, Friedrich A, Müller M, Nolte O, Wolfrum J, Hoheisel JD, Sauer M, Knemeyer J-P (2006)
Nucleic Acids Research 34(13): e90.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Marme, Nicole; Friedrich, Achim; Müller, MatthiasUniBi; Nolte, Oliver; Wolfrum, Jürgen; Hoheisel, Jörg D.; Sauer, MarkusUniBi; Knemeyer, Jens-Peter
Abstract / Bemerkung
We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5' end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10^-8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10^-11 M solutions of PCR amplicons from M.tuberculosis in only 100 s.
Erscheinungsjahr
2006
Zeitschriftentitel
Nucleic Acids Research
Band
34
Ausgabe
13
Seite(n)
e90
ISSN
0305-1048
eISSN
1362-4962
Page URI
https://pub.uni-bielefeld.de/record/1773904

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Marme N, Friedrich A, Müller M, et al. Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy. Nucleic Acids Research. 2006;34(13):e90.
Marme, N., Friedrich, A., Müller, M., Nolte, O., Wolfrum, J., Hoheisel, J. D., Sauer, M., et al. (2006). Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy. Nucleic Acids Research, 34(13), e90. https://doi.org/10.1093/nar/gkl495
Marme, Nicole, Friedrich, Achim, Müller, Matthias, Nolte, Oliver, Wolfrum, Jürgen, Hoheisel, Jörg D., Sauer, Markus, and Knemeyer, Jens-Peter. 2006. “Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy”. Nucleic Acids Research 34 (13): e90.
Marme, N., Friedrich, A., Müller, M., Nolte, O., Wolfrum, J., Hoheisel, J. D., Sauer, M., and Knemeyer, J. - P. (2006). Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy. Nucleic Acids Research 34, e90.
Marme, N., et al., 2006. Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy. Nucleic Acids Research, 34(13), p e90.
N. Marme, et al., “Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy”, Nucleic Acids Research, vol. 34, 2006, pp. e90.
Marme, N., Friedrich, A., Müller, M., Nolte, O., Wolfrum, J., Hoheisel, J.D., Sauer, M., Knemeyer, J.-P.: Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy. Nucleic Acids Research. 34, e90 (2006).
Marme, Nicole, Friedrich, Achim, Müller, Matthias, Nolte, Oliver, Wolfrum, Jürgen, Hoheisel, Jörg D., Sauer, Markus, and Knemeyer, Jens-Peter. “Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy”. Nucleic Acids Research 34.13 (2006): e90.
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