Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency

Authors

  • Priscilla Cukier University of São Paulo; Faculty of Medicine; Hospital das Clínicas
  • Tânia A. S. S. Bachega University of São Paulo; Faculty of Medicine; Hospital das Clínicas
  • Berenice B. Mendonça University of São Paulo; Faculty of Medicine; Hospital das Clínicas
  • Ana Elisa C. Billerbeck University of São Paulo; Faculty of Medicine; Hospital das Clínicas

DOI:

https://doi.org/10.1590/S0041-87812004000600010

Keywords:

Biotin labeling, Large rearrangements, Southern blotting, CAH-21OH

Abstract

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mug), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.

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Published

2004-01-01

Issue

Section

Case Report

How to Cite

Cukier, P., Bachega, T. A. S. S., Mendonça, B. B., & Billerbeck, A. E. C. (2004). Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency . Revista Do Hospital Das Clínicas, 59(6), 369-374. https://doi.org/10.1590/S0041-87812004000600010