Allele-dropout using PCR-based diagnosis for the splicing mutation in intron-2 of the CYP21B-gene: Successful amplification with a TAQ/PWO-polymerasemixture

The splicing mutation in intron 2 (nucleotide 656) of the 21-hydroxylase ge ne (CYP21B) is the most common mutation causing congenital adrenal hyperpla sia (CAH). Homozygosity for nt656G is associated with the classical phenoty pe of CAH. In several studies, a number of clinically asymptomatic relative s of CAM-patients were genotyped as nt656G homozygotes. We have proposed th at the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one allele (in most cases nt656C) during PCR amplification. Here, we report the successful amplification of all alleles at nt656 with a Taq/Pwo DNA polymerase mixture in the primary PCR reaction, The results we re independent from the type of polymerase used for sequencing reactions as the second step in mutation analysis.