Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy.
AUTOR(ES)
Ried, T
RESUMO
Combinatorial labeling of probes (i.e., with two or more different reporters) increases the number of target sequences that can be detected simultaneously by fluorescence in situ hybridization. We have used an epifluorescence microscope equipped with a digital imaging camera and computer software for pseudocoloring and merging images to distinguish up to seven different probes using only three fluorochromes. Chromosome-specific centromere repeat clones and chromosome-specific "composite" probe sets were generated by PCR in which different mixtures of modified nucleotides, including fluorescein-conjugated dUTP, were incorporated. Cosmid clones were labeled similarly by nick-translation. The technique has been used to delineate the centromeres of seven different human chromosomes, on both 4',6-diamidino-2-phenylindole-stained metaphase spreads and interphase nuclei, to map six cosmid clones in a single hybridization experiment and to detect chromosome translocations by chromosome painting. Multiparameter hybridization analysis should facilitate molecular cytogenetics, probe-based pathogen diagnosis, and gene mapping studies.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=48456Documentos Relacionados
- Mapping of single-copy DNA sequences on human chromosomes by in situ hybridization with biotinylated probes: enhancement of detection sensitivity by intensified-fluorescence digital-imaging microscopy.
- Simultaneous visualization of chromosome bands and hybridization signal using colloidal-gold labeling in electron microscopy.
- Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy.
- Fluorescence lifetime imaging by asynchronous pump-probe microscopy.
- Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization