8/23/2023 0 Comments Dot blot for deletion mutationProc Natl Acad Sci U S A 1989 86:6230–6234Ĭai SP, Wall J, Kan YW, Chehab FF: Reverse dot blot probes for the screening of beta-thalassemia mutations in Asians and American blacks. Saiki RK, Walsh PS, Levenson CH, Erlich HA: Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Ordonez-Sanchez ML, Ramirez-Jimenez S, Lopez-Gutierrez AU, et al.: Molecular genetic analysis of patients carrying steroid 21-hydroxylase deficiency in the Mexican population: Identification of possible new mutations and high prevalence of apparent germ-line mutations. Hum Mol Genet 1993 2:499–504ĭay DJ, Speiser PW, Schulze E, et al.: Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees. Wedell A, Luthman H: Steroid 21-hydroxylase deficiency: Two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. White PC, New MI, Dupont B: Structure of human steroid 21-hydroxylase genes. Higashi Y, Tanae A, Inoue H, Hiromasa T, Fujii-Kuriyama Y: Aberrant splicing and missense mutations cause steroid 21-hydroxylase deficiency in humans: Possible gene conversion products. Wedell A: Molecular approaches for the diagnosis of 21-hydroxylase deficiency and congenital adrenal hyperplasia. Strachan T: Molecular pathology of 21-hydrozylase deficiency. Speiser PW, Dupont J, Zhu D, et al.: Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Pang S, Clark A: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Newborn screening and its relationship to the diagnosis and treatment of the disorder. It permits simultaneous detection of a panel of point mutations with only one hybridization per sample and could be automated to study many samples. All mutations were subsequently confirmed by automated DNA sequencing.Ĭonclusion: The RDB method has the advantages of being accurate and cost-effective for the molecular diagnosis of CYP21 point mutations in CAH. Thirty clinically confirmed cases that were identified by the Texas Newborn Screening Program were tested. Signal detection was achieved by chemiluminescence. These exons then were hybridized to membrane strips. DNA was extracted from dried blood spots, and exons encompassing mutations from samples to be tested were amplified and labeled with biotin-dUTP by PCR. Normal and mutant oligonucleotides spanning these nine mutation sites were spotted onto a nylon membrane. Methods and Results: This report is the first application of the reverse dot-blot (RDB) assay for diagnosis of the nine most common point mutations in the CYP21 gene associated with CAH (P30L, g.659A>G or g.659C>G, I172N, I236N-V237E-M239K, V281L, g.l767-1768insT, Q318X, R356W, P453S). Allele-specific PCR, allele-specific oligonucleotide hybridization, and Southern blot analysis are the most common methods to detect point mutations and deletions in the CYP21 gene. Background: More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by mutations of the CYP21 gene that result in deficiencies of the enzyme 21-hydroxylase.
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