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.
Bentolila
Laurent A
LA
University of Chemistry and Biochemistry, California NanoSystems Institute, University of California at Los Angeles, Los Angeles, CA, USA. lbento@chem.ucla.edu
D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002875:Chromosomes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008677:Metaphase; D051379:Mice; D018613:Microscopy, Confocal; D015345:Oligonucleotide Probes / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism*; D055620:Optical Phenomena; D045663:Quantum Dots; D019809:Streptavidin / Q000378:metabolism
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D019809:Streptavidin