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

Endocr Res. 1998 Aug-Nov;24(3-4):637-41. doi: 10.3109/07435809809032662.

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.

MeSH terms

  • Adrenal Hyperplasia, Congenital / genetics*
  • Alleles*
  • DNA, Recombinant*
  • DNA-Directed DNA Polymerase
  • Drug Combinations
  • Female
  • Genotype
  • Heterozygote
  • Homozygote
  • Humans
  • Introns / genetics*
  • Male
  • Mutation / genetics*
  • Polymerase Chain Reaction
  • Steroid 21-Hydroxylase / genetics*
  • Taq Polymerase

Substances

  • DNA, Recombinant
  • Drug Combinations
  • Steroid 21-Hydroxylase
  • Pwo polymerase
  • Taq Polymerase
  • DNA-Directed DNA Polymerase