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Detection of Mutations in the CYP21 Gene Using the DCode System

n some DNA fragments. This was done by PCR-based site-directed mutagenesis.13, 14 The mutant PCR product was mixed with the corresponding wild type PCR product; the mixture was placed at 96 C for 10 minutes to denature the PCR products and subsequently left at room temperature for gradual renaturation, thereby generating heteroduplex molecules.

Denaturing Gradient Gel Electrophoresis
Denaturing gradient gel electrophoresis was carried out using the DCode system (Bio-Rad). Fifteen l of PCR product was loaded on a gel containing a polyacrylamide gradient ranging from 612% and a gradient of urea and formamide. The gels were run in 1x TAE buffer, at 80 V overnight. After electrophoresis, the gels were stained in TAE buffer containing ethidium bromide and subsequently the resolved bands were visualized by ultraviolet (UV) transillumination.


Results
The results of the DGGE analysis of exons 2, 5, 6, and 7 are presented in Figure 1. The presented DGGE analysis was carried out using a denaturing gradient of 3070%. To validate the method, one wild type control sequence and one heterozygous mutant sequence was analyzed for each exon. For exons 2 and 5 the mutant controls were generated by site-directed mutagenesis at the following nucleotide positions: exon 2: 2011 (A→C) and exon 5: 2851 (T→G). Nucleotide positions are given in accordance with CYP21, GenBank: accession numbers M12792; M23280. The mutant control sample for exon 6 contains the cluster-E6 mutation and for exon 7 the Val281Leu mutation.


Discussion
To perform complete genotyping of 21-hydroxylase disease alleles and reliable carrier
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