Flourescence in situ hybridization (FISH) is a diagnostic test that distinguishes chromosomal abnormalities not evident by routine chromosome banding. By tagging and hybridizing defined DNA sequences with fluorochromes, abnormalities can be detected for specific chromosomal loci at much higher resolution (within individual chromosome bands). Commercially available DNA probes, which detect the most common abnormalities, bind to chromosomal targets of 40-1000 kb in length, making them useful for detection of large molecular chromosomal rearrangements. To detect much smaller rearrangements, we have synthesized small single copy (sc) FISH probes designed by genomic sequence analysis (1.5 to 8 kilobases) (Rogan et al, 2001). scFISH permits examination of the chromosome at the resolution of the Southern blot (~2kb). This increased genomic resolution has significant clinical, research and commercial implications, since abnormalities indistinguishable with commercial probes can be classified into distinct categorieswith scFISH probes. However, as a result of their shorter length and reduced chromosomal targets, the fluorescent hybridization signals are, in some instances, smaller than those seen using commercially-available probes.
We aim (1) to develop a web-based application to simplify the selection of scFISH probes throughout the genome and (2) to increase the hybridization signal intensity of small scFISH probes by testing novel DNA labeling/detection technologies and benchmarking them against our currently validated protocol of indirect labeling and detection with fluorophore-conjugated antibodies. We will evaluate two approaches for increasing hybridization signal intensities of hybridized probes with reagents containing either high multiplicity fluorescent adducts (DNA dendrimers) or high quantum yield semicrystalline nanoparticles. The long range goal is to make sc probes as easy to select and visualize as conventional FISH probes so that they can be introduced into clinical cytogenetic laboratories.
| Knoll, Joan H M (2007) Human metaphase chromosome FISH using quantum dot conjugates. Methods Mol Biol 374:55-66 |
