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Detection of Mutations in the CYP21 Gene Using the DCode System

Gita Ohlsson and Marianne Schwartz, Department of Clinical Genetics, Juliane Marie Center, University Hospital, Rigshospitalet, Copenhagen, Denmark


Introduction
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is caused by mutations in the gene CYP21 encoding the enzyme steroid 21-hydroxylase. In addition to deletions, approximately 20 different point mutations have been reported14 and novel mutations are still detected.58 This makes genetic diagnosis as well as carrier detection of 21-hydroxylase deficiency a complicated matter. We have used the denaturing gradient gel electrophoresis method (DGGE) using the DCode universal mutation detection system to detect mutations in the coding sequence and intron-exon junctions of CYP21.9, 10


Materials and Methods
Samples
DNA from healthy individuals and patients with 21-hydroxylase deficiency was isolated from peripheral blood lymphocytes using the salting-out method by Miller et al.11

Polymerase Chain Reaction (PCR)
For amplification of CYP21 and selection against CYP21P, PCR was performed according to Wedell.12 The resulting two PCR products were purified and subsequently used as template for the amplification of DNA fragments for DGGE. The PCR reactions were performed with GC-clamped primers. The GC-density of almost all CYP21 exons are very high requiring long GC-clamps to obtain a single melting domain of the sequence of interest.

Site-Directed Mutagenesis
In order to obtain mutant control samples for all exons it was necessary to introduce mutations i
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