Different donors should be tested as hybridization may be variable. Commercially available slides may be used, but should always be tested first. Isolation of DNA from test tissue and reference tissue Standard phenol extraction is used to obtain DNA from test or reference (karyotypically normal individual) tissue, which involves the combination of Tris-Ethylenediaminetetraacetic acid and phenol with aqueous DNA in equal amounts. This is followed by separation by agitation and centrifugation, after which the aqueous layer is removed and further treated using ether and finally ethanol precipitation is used to concentrate the DNA. May be completed using DNA isolation kits available commercially which are based on affinity columns. Preferentially, DNA should be extracted from fresh or frozen tissue as this will be of the highest quality, though it is now possible to use archival material which is formalin fixed or paraffin wax embedded, provided the appropriate procedures are followed. 0.5-1 μg of DNA is sufficient for the CGH experiment, though if the desired amount is not obtained DOP-PCR may be applied to amplify the DNA, however it in this case it is important to apply DOP-PCR to both the test and reference DNA samples to improve reliability. DNA labelling Nick translation is used to label the DNA and involves cutting DNA and substituting nucleotides labelled with fluorophores (direct labelling) or biotin or oxigenin to have fluophore conjugated antibodies added later (indirect labelling). It is then important to check fragment lengths of both test and reference DNA by gel electrophoresis, as they should be within the range of 500kb-1500kb for optimum hybridization. Blocking Unlabelled Life Technologies Corporation's Cot-1 DNA (placental DNA enriched with repetitive sequences of length 50bp-100bp)is added to block normal repetitive DNA sequences, particularly at centromeres and telomeres, as these sequences, if detected, may reduce the fluorescence ratio and cause gains or losses to escape detection. Hybridization 8–12μl of each of labelled test and labelled reference DNA are mixed and 40 μg Cot-1 DNA is added, then precipitated and subsequently dissolved in 6μl of hybridization mix, which contains 50% formamide to decrease DNA melting temperature and 10% dextran sulphate to increase the effective probe concentration in a saline sodium citrate (SSC) solution at a pH of 7.0. Denaturation of the slide and probes are carried out separately. The slide is submerged in 70% formamide/2xSSC for 5–10 minutes at 72 °C, while the probes are denatured by immersion in a