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

y, to generate heteroduplexes between heterozygous PAH gene fragments, the PCR fragments were incubated for 5 minutes at 95 C, 60 minutes at 65 C and 60 minutes at 37 C. Complete amplification mixes were used for DGGE analysis.

MP-DGGE was performed under two different electrophoresis conditions. A 6% polyacrylamide gel containing a 2070% (for MP2, MP3 and MP10) or a 3080% (for MP1 and MP7) gradient (Model 475 Gradient Delivery system, Bio-Rad) of urea and formamide (100% is 7 M urea and 40% formamide) was run at 60 C in TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). Electrophoresis conditions were 130 V for 6 hours on the DCode apparatus (Bio-Rad). Gels were stained in 1 g/ml ethidium bromide and visualized by UV transillumination.

The point mutations were confirmed by sequencing the PCR amplified fragments with the non-GC clamped primer of each exon, using the Applied Biosystems Model 373A DNA Sequencing System and the PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems).


Results

Denaturing gradient gel electrophoresis allows detection of more than 95% of the PKU causing mutations. In the Belgian population, we observed about 100 different mutations using DGGE technology. Among these, seven new mutations were identified (Michiels et al.,1998).4 The high mutation resolving power of DGGE is illustrated in Figure 1. This figure shows all the mutations or mutation combinations we located in exon 7 of Belgian PKU patients. The screening of PKU patients and their relatives is rather laborious when exon specific amplification and DGGE conditions are used. Therefore, to obta
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