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

• Autor(a) principal: Cukier,Priscilla
• Data de Publicação: 2004
• Outros Autores: Bachega,Tânia A. S. S., Mendonça,Berenice B., Billerbeck,Ana Elisa C.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Revista do Hospital das Clínicas
• Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0041-87812004000600010
• topic: Biotin labeling; Large rearrangements; Southern blotting; CAH-21OH

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.