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).

Download
OA
Journal Article | Published | English
Author
; ; ; ; ; ; ;
Abstract
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.
Publishing Year
ISSN
eISSN
PUB-ID

Cite this

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).
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).
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.
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).
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.
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, (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).
Main File(s)
File Name
Access Level
OA Open Access

This data publication is cited in the following publications:
This publication cites the following data publications:

5 Citations in Europe PMC

Data provided by Europe PubMed Central.

Tunable blinking kinetics of cy5 for precise DNA quantification and single-nucleotide difference detection.
Yeh HC, Puleo CM, Ho YP, Bailey VJ, Lim TC, Liu K, Wang TH., Biophys. J. 95(2), 2008
PMID: 18424494

41 References

Data provided by Europe PubMed Central.

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy.
Kim J, Doose S, Neuweiler H, Sauer M., Nucleic Acids Res. 34(9), 2006
PMID: 16687657
Frequency of rpoB Mutations Inside and Outside the Clusters I Region in Rifampicin-Resistant Isolates
Heep M., Brandstatter B., Rieger U., Lehn N., Richter E., Rusch-Gerdes S., Niemann S.., 2000
Characterization of rifampicin-resistant Mycobacterium tuberculosis in Taiwan.
Hwang HY, Chang CY, Chang LL, Chang SF, Chang YH, Chen YJ., J. Med. Microbiol. 52(Pt 3), 2003
PMID: 12621089
Relationship between antimycobacterial activities of rifampicin, rifabutin and KRM-1648 and rpoB mutations of Mycobacterium tuberculosis.
Yang B, Koga H, Ohno H, Ogawa K, Fukuda M, Hirakata Y, Maesaki S, Tomono K, Tashiro T, Kohno S., J. Antimicrob. Chemother. 42(5), 1998
PMID: 9848446

Zuker M., Mathews D.H., Turner D.H.., 1999
Photophysical dynamics of single dye molecules studied by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM)
Tinnefeld P., Herten D.P., Sauer M.., 2001
Bias and precision in the estimation of exponential decay parameters from sparse data
Tellinghuisen J., Wilkerson C.W.., 1993
Error analysis of simple algorithms for determining fluorescence lifetimes in ultradilute dye solutions
Soper S.A., Legendre B.L.., 1994
Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy
Magde D., Elson E.L., Webb W.W.., 1972
Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy
Widengren J., Schwille P.., 2000
Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy: theory and experimental realizations
Widengren J., Schweinberger E., Berger S., Seidel C.A.M.., 2001
Autofluorescence of viable cultured mammalian cells
Aubin J.E.., 1979
Reduction of luminescent background in ultrasensitive fluorescence detection by photobleaching
Affleck R.L., Ambrose W.P., Demas J.N., Goodwin P.M., Schecker J.A., Wu M., Keller R.A.., 1996
Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers
Sauer M., Zander C., Müller R., Ullrich B., Drexhage K.H., Kaul S., Wolfrum J.., 1997
Branching out of single-molecule fluorescence spectroscopy: challenges for chemistry and influence on biology
Tinnefeld P., Sauer M.., 2005

Export

0 Marked Publications

Open Data PUB

Web of Science

View record in Web of Science®

Sources

PMID: 16870719
PubMed | Europe PMC

Search this title in

Google Scholar