Gene dosage of GLI was analysed by Fluorescent Differential PCR employing FAM labelled oligonucleotide primers [18 (link)]. Two reference loci were also used: exon 19 of the cystic fibrosis transmembrane conductance regulator (CFTR) at 7q31 and ubiquitin-conjugating enzyme pseudogene (UBE2L2) at 12q12. PCR amplification was carried out in 30 μl 10 mM Tris-HCl buffer, pH 9.0, containing 50 mM KCl, 0.1% (v/v) Triton X-100, 1.5 mM MgCl2, 0.2 mM dNTPs, 5 μM of each primer for UBE2L2 and CFTR, 100 ng of genomic DNA, 7.5 μM of the GLI primers and 2 units Taq DNA polymerase. PCR conditions consisted of an initial denaturation step of 95°C for 5 minutes, followed by 24 cycles of 95°C for 1 minute, 58°C for 1 minute and 72°C for 1 minute, with a final extension of 72°C for 10 minutes. A PCR using human genomic DNA as a control was routinely run in parallel. Moreover, experiments were performed in triplicate to ensure reproducibility of the technique. The FAM labelled products were separated through a denaturing polyacrylamide gel on an ABI 377 fragment analyser (PE Applied Biosystems). Quantitative analysis of the PCR products was performed using Genescan 672 software© (PE Applied Biosystems). GLI gene dosage was calculated by comparing the ratios of GLI to reference gene peak areas generated by control DNA with those generated by the cell line DNAs, as described [18 (link)]. Primer sequences were as follows: GLI-F-d TGA TGC AGT TCC TTT ATT ATC AGG; GLI-R-(FAM)-d AGA GTA GGG AAT CTC ATC CAT CA, giving a product of 200 bp. UB-F(FAM)-d CGA AGA GCA CAC TTA AAG ATC TG; Ub-R-d GGT CAG CCT-GAA GTG GAT GCT CA generating a 173 bp product. CFTR19-F-dCCT ACC AAG TCA ACC AAA CC; CFTR19-R(FAM)-dACA TTG CTT CAG GCT ACT GG; generating a product of 268 bp.