The coding region of Cx32 was amplified by touchdown polymerase chain reaction (PCR) (Don et al., 1991). Three primer sets were used corresponding to nucleotides 54-77 (CX32-1) and 336-359 (CX32-2), i.e., part 1, 273-269 (Cx32-3) and 685-704 (Cx32-5), i.e., part 2, and 635-658 (Cx32-S1) and 919-938 (Cx32-A1), i.e., part 3 (primer sequences from Bergoffen et al., 1993). A schematic representation of the amplification fragments is given in figure 1. The reaction was carried out in a 25-ãl volume containing 100 ng genomic DNA template and 25 pmole of each oligodeoxynucleotide primer and in case of radioactive SSCP, 0.1 ãl [~-32 P]dCTP (Amersham, Buckinghamshire, UK). The PCR conditions were as follows: denaturation at 94°C for 90", followed by 40 cycles of 60" at 94°C, 90" at an annealing temperature decreasing from 70°C to 55°C (decrease of 1°C each 2 cycles, 10 cycles at 55°C), 120" at 12°C and a final extension step of 5´ at 72°C. Samples of amplified DNA were mixed with formamide sample buffer and after denaturation for 2' at 100°C and rapid cooling on ice subjected to electrophoresis on a 1 × HydroLink MDE (J.T. Baker, Phillipsburg, NY) gel with and without 10% glycerol at 15 W for 18-20 hr, allowing the detection of known Cx32 mutations. As positive controls in the SSCP analysis we used DNA samples from patients with following Cx32 mutations: R215W (Fairweather et