Fluorescence in situ Hybridization (FISH) pp 147-163
Direct In Situ Hybridization with Oligonucleotide Functionalized Quantum Dot Probes
Coming from the material sciences, fluorescent semiconductor nanocrystals, also known as quantum dots (QDs), have emerged as powerful fluorescent probes for a wide range of biological imaging applications. QDs have several advantages over organic dyes which include higher brightness, better resistance to photobleaching, and simplified multicolor target detection. In this chapter, we describe a rapid assay for the direct imaging of multiple repetitive subnuclear genetic sequences using QD-based FISH probes. Streptavidin-coated QDs (SAvQDs) are functionalized with short biotinylated oligonucleotides and used in a single hybridization/detection step. These QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and show good targeting of dense chromatin domains. Importantly, the broad absorption spectra of QDs allows two sequence specific QD-FISH probes of different colors to be simultaneously imaged with a single laser excitation wavelength. This method, which requires minimal custom conjugation, is easily expandable and offers the experimentalist a new alternative to increase flexibility in multicolor cytogenetic FISH applications of repetitive DNAs.
Key wordsBiomaterial Chromosome Confocal laser scanning microscopy Cytogenetic FISH Fluorescence Hybridization Imaging Multiplex labeling Nanotechnology Oligonucleotide Quantum dot Repetitive DNA
- 10.Bentolila, L. A., Michalet X., and Weiss S. (2008) Quantum optics: colloidal fluorescent semiconductor nanocrystals (quantum dots) in single-molecule detection and imaging. In: Single-Molecules and Biotechnology, (Rigler, R. and Vogel, E., Eds), Springer, Berlin, Heidelberg, pp. 53–81.CrossRefGoogle Scholar
- 14.Rattray, M. and Michael, G. J. (1998) Oligonucleotide probes for in situ hybridization. In: In Situ Hybridization (Wilkinson, D. G., Ed.), Oxford University Press, Oxford, pp. 23–67.Google Scholar