Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy.
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.
Marmé
Nicole
N
Institute of Physical Chemistry, University of Heidelberg Im Neuenheimer Feld, 229, 69120 Heidelberg, Germany.
Friedrich
Achim
A
Müller
Matthias
M
Nolte
Oliver
O
Wolfrum
Jürgen
J
Hoheisel
Jörg D
JD
Sauer
Markus
M
Knemeyer
Jens-Peter
JP
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D005456:Fluorescent Dyes / Q000737:chemistry*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012293:Rifampin / Q000494:pharmacology; D017422:Sequence Analysis, DNA / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin