Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) improves signal intensity and increases rRNA accessibility.
Fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5'-, 3'-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase in situ accessibility of rRNA target sites and thus provide more flexibility for probe design.
Stoecker
Kilian
K
Department of Microbial Ecology, University of Vienna, Vienna, Austria.
Dorninger
Christiane
C
Daims
Holger
H
Wagner
Michael
M
D017124:Conserved Sequence / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry*; D010802:Phylogeny; D012329:RNA, Bacterial / Q000032:analysis*; D012335:RNA, Ribosomal / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D016415:Sequence Alignment; D013045:Species Specificity; D013816:Thermodynamics
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012337:RNA, Ribosomal, 18S