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Identification of Disease Causing Mutations in Phenylketonuria by Denaturing Gradient Gel Electrophoresis Using the DCode System

> 100 extraction procedure (Walsh et al., 1991).5 Alternatively 0.5 ml fresh EDTA-treated blood sample was treated with 1 ml haemolysis buffer (HB) at 4 C for 10 minutes. White blood cells were pelleted (5 minutes at 2,500 rpm in a microfuge) and washed with HB until no red cell debris was left. After the removal of the supernatant, 500 l of 5% Chelex 100 (Bio-Rad, California) suspension in water was added and mixed thoroughly.

This step was followed by incubations at 56 C for 30 minutes and subsequently at 100 C for 5 minutes. After centrifuging (3 minutes at 2,500 rpm), the supernatant was stored at -20 C. Five l of these DNA preparations was used in a 20 l multiplex amplification reaction of the PAH exons and their intron-exon boundaries. GC-clamped primers for DGGE analysis of the PAH gene have been described by Guldberg et al., 1993.1 To achieve comparable PCR amplification efficiencies and non-overlapping, but high mutation resolution DGGE patterns, combinations of these primer sets were evaluated (Michiels et al. 1996).3 Amplifications were carried out in 20 l reaction mixtures, containing Perkin Elmer PCR reaction buffer with 1.5 mM MgCl2, 200 nM each dNTP, 800 nM each primer (MP 1: exon 1 + exon 4 + exon 13, MP2: exon 2 + exon 6+ exon 8, MP3 : exon 3 + exon 5 + exon 9, MP7 : exon 7 + exon 11, MP10: exon 10 + exon 12.) and 1.5 U of Taq DNA polymerase (Perkin Elmer). The Perkin Elmer 9600 thermal cycler was set at 5 minutes denaturation at 95 C followed by 40 cycles of 10 seconds denaturation at 95 C, 20 seconds annealing at 56 C and 40 seconds elongation at 72 C. After cycling, final elongation was done at 72 C for 5 minutes. Finall
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