Fluorescence in situ Hybridization (FISH) pp 147-163

Part of the Methods in Molecular Biology book series (MIMB, volume 659)

Direct In Situ Hybridization with Oligonucleotide Functionalized Quantum Dot Probes

Protocol

Abstract

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 words

Biomaterial Chromosome Confocal laser scanning microscopy Cytogenetic FISH Fluorescence Hybridization Imaging Multiplex labeling Nanotechnology Oligonucleotide Quantum dot Repetitive DNA 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.University of Chemistry and Biochemistry, California NanoSystems InstituteUniversity of California at Los AngelesLos AngelesUSA

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