As a clinical cytogeneticist engaging in interpretation of FISH data (Fluorescence in situ Hybridization) every day, I often find myself wondering the rationale behind probe design and the roles that clinical cytogeneticists played in this process. Take, for example, the case of the BCL2 gene, which can occasionally be inserted into the IGH locus or other genes’ loci. The break-apart (BA) probes provided by leading FISH probe suppliers fail to encompass the BCL2 gene locus (Fig B, right), therefore, they are unable to detect such gene insertions. This limitation proves particularly troublesome when only MYC, BCL2, and BCL6 BA probes are included in the high-grade B-cell lymphoma (HGBL) FISH panel. In such scenarios, if a patient harbors both a typical MYC rearrangement and a BCL2 insertion into the IGH (or IGK or IGL) locus, the FISH analysis would only show the presence of the MYC rearrangement, potentially overlooking the diagnosis of HGBL involving both MYC and BCL2 rearrangements.
To address this gap, we introduced BRIGHTDOM BCL2 break-apart probes, specifically labeling the BCL2 gene locus with a red fluorescent dye (Fig. A, right). In Case 1, we observed the insertion of the BCL2 gene into the IGH gene locus (Fig. C, right), while Case 2 presented an insertion into a locus other than IGH. Notably, in both instances, the BRIGHTDOM BCL2 BA probes exhibited an abnormal signal pattern, offering valuable insights into these complex genomic rearrangements.
All BRIGHTDOM’S break-apart probes cover the functionally critical regions, e.g. the whole genes such as MYC, BCL2, CCND1…. or the functional domains of the fusion genes, such as 3’ ABL1, 3’ ABL2… This is especially helpful when we interpret a complex FISH signal pattern.
The BRIGHTDOM Sequence-specific Single–stranded DNA FISH Probes
The regions of interest are selected based on recent peer-reviewed literature and the latest human genome database (GRCh38). All the probes are designed in silico, eliminating the limitations of availability of bacterial artificial clones (BAC). The resolution of the breakpoints is accurate down to the single base level.
The BRIGHTDOM Probes only target the specific sequence within the regions of interest, minimizing the cross hybridization and reducing the background signals.
The BRIGHTDOM Probes are mixtures of fluorescence-labeled signal-stranded and double stranded probes. The single-stranded probes greatly increase the binding efficiency and significantly reduce hybridization time. Both strands of the double-stranded probes are fluorescence-labeled, therefore, either of the strands will give fluorescent signals when they reach the target regions.
The BRIGHTDOM Probes and the BRIGHTDOM H2 fast hybridization buffer enable the clinical laboratories to finish FISH wet-lab process (from dropping FISH slide to getting FISH slide ready for analysis) in less than three hours.