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

in compatible co-amplifications with comparable efficiencies, different exon PCR amplifications were combined. Moreover, to make sure that the resolving power to identify the exon specific mutations remains intact and that the DGGE patterns of the combined exons do not interfere with each other, the multiplex PCR amplifications have to be resolved subsequently on denaturing gradient gels. This resulted in a multiplex DGGE (MP-DGGE) of the PAH gene (Michiels et al. 1996).3 Examples of such an MP-DGGE are shown in Figure 2. The 13 different PAH exons are analyzed in 5 distinct multiplex PCR and DGGE patterns. Each exon shows distinct mutations, demonstrating that the resolving power of the DGGE gel remains intact. In addition, the DGGE patterns for the different exons do not overlap. Cycle sequencing of the DGGE DNA bands excised from the gel as described in the methods allows the identification of new mutations.

Several DNA preparation protocols have been tested: home made (see Methods) or commercially available kits such as Split Second from Boehringer Mannheim, Germany and DNA Easy-Prep from Lifecodes Corporation, USA, and can all be used in the MP-DGGE assay. All of them gave comparable results in the multiplex PCR/DGGE experiments.


Conclusion

In this report we demonstrate the use of powerful multiplex PCR amplification combined with DGGE analysis to rapidly identify mutations causing PKU. This MP-DGGE allows analysis of the complete PAH gene for three different individuals on one gel, whereas previously such analysis would take three gel runs. Moreover, the multiplex DGGE analysis is compatible with all the tested rapid DNA iso
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