RAPID DETECTION OF 21-HYDROXYLASE DEFICIENCY MUTATIONS BY ALLELE-SPECIFIC IN-VITRO AMPLIFICATION AND CAPILLARY-ZONE-ELECTROPHORESIS

A quick diagnosis of the classic form of 21-hydroxylase deficiency (si mple virilizing and salt wasting) is of great importance, especially f or prenatal diagnosis and treatment in pregnancies at risk. A method f or simultaneous detection of common point mutations in the P450c21 B g ene is here proposed by combining a nested PCR amplification refractor y mutation system (ARMS) with capillary zone electrophoresis (CZE) in sieving liquid polymers. In the first PCR, B genes are selectively amp lified. In the nested reaction, ARMS-detected wildtype and mutated all eles are separately pooled and resolved by CZE. CZE is performed in co ated capillaries in the presence of 30 g/L hydroxyethyl cellulose in t he background electrolyte for size separation of the DNA analytes. For high-sensitivity detection the electrophoresis buffer contains the fl uorescent dye SYBR Green I. laser-induced fluorescence detection is ob tained by excitation at 488 nm and signal collection at 520 nm. Specif icity and reproducibility of the protocols were established by using s amples from 75 Italian families with 21-hydroxylase deficiency already genotyped by allele-specific oligonucleotide hybridization or direct sequencing. Whereas dot-blot is time consuming because of the high num ber of hybridizations with radioactive probes, this present protocol i s more rapid, giving sufficient separation on CZE after PCR reactions without preconcentration or desalting of samples.