Comparative genomic hybridisation (CGH)
Chromosomal CGH (cCGH) is a robust screening technique based on competitive hybridization of tumor and normal DNA, labeled with different fluorochromes, onto normal metaphase spreads. This methodology detects genomic imbalances in tumor cells (specific DNA gains and/or losses within chromosomes) based on the fluorescent ratio measured after hybridization, providing a practical whole-genome analysis in a single test. The main advantage of the procedure, in contrast to chromosomal banding techniques, is the fact that it does not require cell culturing (tumor DNA can be extracted from fresh or archival tissue samples). The two main downsides relate to the inability in disclosing balanced chromosome rearrangements (genetic events that do not result in gain or loss of chromosomal material) and the lower resolution potential (estimates vary from 3 to 10 Mb depending on the aberration in question), which makes it necessary to follow up on results with a more selective analysis. As most carcinomas are known to harbor complex genetic aberrations resulting in gain and loss of genetic material, however, cCGH emerges as an excellent approach for their detection, and high-throughput methodologies have been established that can rapidly validate and extend the findings.
Method Overview
Sample DNA (tumor) and reference DNA (peripheral blood lymphocytes from a donor) is extracted using standard procedures and labeled in a nick translation reaction with two different fluorochromes. The same amount of labeled test and reference DNA is then mixed (with the addition of Cot-1 DNA to repress repetitive sequences), precipitated, dried, and dissolved in hybridization buffer. This probe mixture is then denatured and hybridized to commercially available, normal metaphase slides for 2-3 days at 37 ºC in a moist chamber. After washing off excess probe, samples are counterstained and the single-color images corresponding to DAPI, green, and red fluorochrome hybridization signals are sequentially captured using a fluorescence microscope. For each sample, ten high-quality metaphase spreads are selected for analysis. Chromosomes are identified based on their inverted DAPI appearance and the relative signal intensity is determined along each chromosome. Data from the 10 cells is then combined to generate an average profile with 99 % confidence intervals for that sample. For the scoring of cCGH results we have recently adopted the use of dynamic standard reference intervals (D-SRI), which have been shown to provide a more objective and sensitive scoring criterion when compared to fixed thresholds. Our current D-SRI (generated with data from 10 normal vs normal hybridizations) is automatically scaled onto each sample profile and aberrations are scored whenever the case profile and the standard reference profile at 99 % confidence do not overlap.
At present, cCGH is being performed both at the Lothe lab and at the Teixeira lab, using the same protocol, quality controls, and scoring criteria. A joint database has been created that includes the genomic information obtained from the samples analyzed in both laboratories.
Method Overview
Sample DNA (tumor) and reference DNA (peripheral blood lymphocytes from a donor) is extracted using standard procedures and labeled in a nick translation reaction with two different fluorochromes. The same amount of labeled test and reference DNA is then mixed (with the addition of Cot-1 DNA to repress repetitive sequences), precipitated, dried, and dissolved in hybridization buffer. This probe mixture is then denatured and hybridized to commercially available, normal metaphase slides for 2-3 days at 37 ºC in a moist chamber. After washing off excess probe, samples are counterstained and the single-color images corresponding to DAPI, green, and red fluorochrome hybridization signals are sequentially captured using a fluorescence microscope. For each sample, ten high-quality metaphase spreads are selected for analysis. Chromosomes are identified based on their inverted DAPI appearance and the relative signal intensity is determined along each chromosome. Data from the 10 cells is then combined to generate an average profile with 99 % confidence intervals for that sample. For the scoring of cCGH results we have recently adopted the use of dynamic standard reference intervals (D-SRI), which have been shown to provide a more objective and sensitive scoring criterion when compared to fixed thresholds. Our current D-SRI (generated with data from 10 normal vs normal hybridizations) is automatically scaled onto each sample profile and aberrations are scored whenever the case profile and the standard reference profile at 99 % confidence do not overlap.
At present, cCGH is being performed both at the Lothe lab and at the Teixeira lab, using the same protocol, quality controls, and scoring criteria. A joint database has been created that includes the genomic information obtained from the samples analyzed in both laboratories.