Quantum dots as new-generation fluorochromes for FISH: an appraisal.
In the field of nanotechnology, quantum dots (QDs) are a novel class of inorganic fluorochromes composed of nanometre-scale crystals made of a semiconductor material. Given the remarkable optical properties that they possess, they have been proposed as an ideal material for use in fluorescent in-situ hybridization (FISH). That is, they are resistant to photobleaching and they excite at a wide range of wavelengths but emit light in a very narrow band that can be controlled by particle size and thus have the potential for multiplexing experiments. The principal aim of this study was to compare the potential of QDs against traditional organic fluorochromes in both indirect (i.e. QD-conjugated streptavidin) and direct (i.e. synthesis of QD-labelled FISH probes) detection methods. In general, the indirect experiments met with a degree of success, with FISH applications demonstrated for chromosome painting, BAC mapping and use of oligonucleotide probes on human and avian chromosomes/nuclei. Many of the reported properties of QDs (e.g. brightness, 'blinking' and resistance to photobleaching) were observed. On the other hand, signals were more frequently observed where the chromatin was less condensed (e.g. around the periphery of the chromosome or in the interphase nucleus) and significant bleed-through to other filters was apparent (despite the reported narrow emission spectra). Most importantly, experimental success was intermittent (sometimes even in identical, parallel experiments) making attempts to improve reliability difficult. Experimentation with direct labelling showed evidence of the generation of QD-DNA constructs but no successful FISH experiments. We conclude that QDs are not, in their current form, suitable materials for FISH because of the lack of reproducibility of the experiments; we speculate why this might be the case and look forward to the possibility of nanotechnology forming the basis of future molecular cytogenetic applications.
Ioannou
Dimitris
D
Department of Biosciences, University of Kent, Canterbury, UK.
Tempest
Helen G
HG
Skinner
Benjamin M
BM
Thornhill
Alan R
AR
Ellis
Michael
M
Griffin
Darren K
DK
D000818:Animals; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002232:Carbocyanines / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D020223:Chromosome Painting; D002875:Chromosomes / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D002881:Chromosomes, Human, Pair 12 / Q000235:genetics; D002999:Clone Cells; D004247:DNA / Q000378:metabolism; D004076:Digoxigenin / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006824:Hybridization, Genetic; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007202:Indicators and Reagents / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D008297:Male; D008677:Metaphase; D008856:Microscopy, Fluorescence; D036103:Nanotechnology / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D038761:Photobleaching; D045663:Quantum Dots; D012666:Semiconductors; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007211:Indoles; D015345:Oligonucleotide Probes; D014966:Xanthenes; C089187:cyanine dye 3; C007293:DAPI; D001710:Biotin; C034657:Texas red; D004247:DNA; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate; D004076:Digoxigenin