title: Allele-dropout using PCR-based diagnosis for the splicing mutation in intron-2 of the CYP21B-gene: successful amplification with a Taq/Pwo-polymerase mixture.
abstract: The splicing mutation in intron 2 (nucleotide 656) of the 21-hydroxylase gene (CYP21B) is the most common mutation causing congenital adrenal hyperplasia (CAH). Homozygosity for nt656G is associated with the classical phenotype of CAH. In several studies, a number of clinically asymptomatic relatives of CAH-patients were genotyped as nt656G homozygotes. We have proposed that 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 were independent from the type of polymerase used for sequencing reactions as the second step in mutation analysis.
authors.affiliation: Department of Pharmacology, University of Heidelberg, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Schulze
authors.forename: M
authors.initials: M
authors.lastname: Bettendorf
authors.forename: C
authors.initials: C
authors.lastname: Maser-Gluth
authors.forename: M
authors.initials: M
authors.lastname: Decker
authors.forename: U
authors.initials: U
authors.lastname: Schwabe
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics*; D000483:Alleles; D004274:DNA, Recombinant; D004259:DNA-Directed DNA Polymerase; D004338:Drug Combinations; D005260:Female; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007438:Introns / Q000235:genetics*; D008297:Male; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013255:Steroid 21-Hydroxylase / Q000235:genetics*; D019914:Taq Polymerase
chemical_list: D004274:DNA, Recombinant; D004338:Drug Combinations; D013255:Steroid 21-Hydroxylase; C118225:Pwo polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase