Abstract
In humans two 21-OH genes with high homology are present: A (inactive) and B (active). A modification of PCR which amplifies specific alleles, would allow for specific amplification of the active gene. The purpose of the present study was to develop a PCR technique for detection of partial or total cP450 21B gene deletions or gene conversions. These abnormalities accomit for 25% reported abnormalities in other populations. DNA template was extracted from peripheral leucocytes (3ml blood samples). For PCR 500 ng template, 2.5 U Taq I polymerase, 4mM MgCl2 and 0.2uM of 2 oligonucleotides (primers) were mixed. These primers will amplify 300 pB of Exon 3, Intro 3 and a fraction of Exon 4 for cP450 21B. Thirty five cycles (denaturation 1 min at 93° C, hybridization 2 min at 60° C and extension 3 min at 72° C) were carried out. Amplification of B-globin gene was used as control. The amplified product was analyzed by 1% agarose gel electrophoresis with ethidium bromide in the presence of molecular weight standards. Ten patients with CAH (6 salt losers) and ten normal control (C) were studied. cP450 21B was amplified in 9/10 patients and in the 10 C. The patient in whom cP450 21B was not amplified, the B-globin was normally amplified. This patient was a salt loser. It is concluded that the method can show partial or total gene deletions, as well as gene conversions, in 21-OH deficiency. Point mutations probably require restriction enzymes analysis of the amplified product.
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Dardis, A., Belgorosky, A. 3 DIRECT ANALYSIS OF THE cP450 21B GENE IN 21-HYDROXYLASE DEFICIENCY (21-OH) USING THE POLYMERASE CHAIN REACTION (PCR). Pediatr Res 36, 672 (1994). https://doi.org/10.1203/00006450-199411000-00027
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DOI: https://doi.org/10.1203/00006450-199411000-00027