Published online 26 July 2006
Nucleic Acids Research, 2006, Vol. 34, No. 13 e90
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy
1 Institute of Physical Chemistry, University of Heidelberg Im Neuenheimer Feld 229, 69120 Heidelberg, Germany 2 Department of Functional Genome Analysis, German Cancer Research Center Im Neuenheimer Feld 580, 69120 Heidelberg, Germany 3 Institute of Physical Chemistry, University of Heidelberg Im Neuenheimer Feld 253, 69120 Heidelberg, Germany 4 Applied Laser Physics and Laser Spectroscopy, University of Bielefeld Universitätsstrasse 25, 33615 Bielefeld, Germany
*To whom correspondence should be addressed. Tel: +49 622 154 5044; Fax: +49 622 154 5050; Email: knemeyer{at}single-molecule-spectroscopy.de
Received May 22, 2006. Revised June 28, 2006. Accepted June 29, 2006.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors