GTG banding and FISH were carried out on metaphases derived from short term lymphocyte cultures stimulated with phytohaemagglutinin M by standard techniques. Probes for whole chromosome paints (1, 2, 7, 8, 11, 16, and 17), SNRPN, D3S1442, D1S1615, and the 11p telomere (Tel 11p) were obtained from Oncor and hybridised under conditions recommended by the supplier. Whole chromosome paints for chromosomes 9 and 20 were obtained from Cytocell and hybridised under recommended conditions.

DNA from test and reference samples was carefully quantified using a fluorimeter with known standards. Exactly 1 μg of test DNA from a patient with an unbalanced karyotype was labelled with Spectrum green-dUTP (Vysis) and 1 μg of reference (normal) DNA with Spectrum red-dUTP(Vysis) using a CGH nick translation kit from Vysis. Ten μl of nick translation enzyme (DNAase and polymerase I) from this kit were used for each nick translation reaction carried out at 15°C for four hours. An aliquot of each reaction was run on a 1% agarose gel to determine the probe size, the optimum size required being 300 bp to 3 kb. Test and reference DNA were then ethanol precipitated in the presence of 50 × human Cot-1 DNA (to block highly repetitive sequences), and resuspended in 10 μl of hybridisation buffer (50% formamide, 1 × SSC, 10% dextran sulphate) overnight at 37°C to aid resuspension.

Hybridisation was performed on commercially prepared (Vysis) slides containing metaphases. The metaphase DNA was denatured by immersing slides in 70% formamide, 2 × SSC at 73°C for five minutes.

The slides were then dehydrated in an ethanol series (70%, 85%, and 100%) and air dried. The probe DNA was denatured at 73°C for five minutes and added immediately to the slides. Slides were coverslipped, sealed with rubber cement, and placed in a moist container at 37°C for 72 hours.

Unbound DNA fragments were removed by washing in 0.4 × SSC/0.3% NP-40 at 73°C for two minutes and 2 × SSC/0.1% NP-40 at room temperature for 30 seconds. Slides were then counterstained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI-

antifade from Oncor).

Separate digitised grey level images of DAPI, Spectrum green, and Spectrum red fluorescence were acquired with a CCD camera coupled to an Olympus BX60 microscope. Image processing was carried out using Cytovision 3.52 software from Applied Imaging. Average green:red fluorescence ratio profiles were calculated for each chromosome from 8-10 metaphases. As an internal control, each CGH experiment was performed by mismatching the sexes between test and reference DNA. If the green labelled test DNA is male (single X chromosome) and the red labelled reference DNA is female (two X chromosomes), then we would expect a green to red fluorescence ratio (FR) on the target X chromosome of 0.5. For the converse situation where the green labelled test DNA is female and the red labelled reference DNA is male, we would expect a green to red fluorescence ratio (FR) on the target X chromosome of 2.0. Significant deviation from these theoretical FRs indicates a poor CGH experiment.

Abnormalities on the X chromosome can still be observed using FR values of either 0.5 or 2.0 as baselines.

Last Updated on: Nov 27, 2024