Protocols
ChromoTrax Biomedical probes are produced by PCR ampliflication of microdissected regions of normal human chromosomes. Template DNA for each probe is amplified using biotin-labeled dUTP and the UN1 primer. The UN1 primer is designed to avoid human repetitive elements. This is particularly important for the terminal band probes in which telomeric repeats are excluded as a result of this amplification method. The resultant specific probes can be used in any FISH protocol of the researchers choice, but we recommend the ChromoTrax Biomedical protocol as a good starting point.
The ChromoTrax Biomedical FISH protocol for biotin-labeled probes relies on a 'sandwich' technique for immuno amplification of signal. Briefly, following hybridization, the biotin-labeled probe is incubated with fluoroscein-labeled avidin. For most of our probes, the probe can be directly viewed after washing the slide. However, we recommend that the rest of the amplification steps be followed. These include washing the slide and incubating with biotin-labeled anti-avidin. This antibody binds to the avidin that is already complexed to the biotin in the probe. After washing the probe, a second round of fluoroscein-labeled avidin is used which complexes with the biotin attached to the anit-avidin. This amplification procedure ensures easy signal visualization, even under conditions where weaker primary signals or poor hybridization exist.