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 (simple virilizing and salt wasting) is of great importance, especially for prenatal diagnosis and treatment in pregnancies at risk. A method for simultaneous detection of common point mutations in the P450c21 B gene is here proposed by combining a nested PCR amplification refractory mutation system (ARMS) with capillary zone electrophoresis (CZE) in sieving liquid polymers. In the first PCR, B genes are selectively amplified. In the nested reaction, ARMS-detected wild-type and mutated alleles are separately pooled and resolved by CZE. CZE is performed in coated capillaries in the presence of 30 g/L hydroxyethyl cellulose in the background electrolyte for size separation of the DNA analytes. For high-sensitivity detection the electrophoresis buffer contains the fluorescent dye SYBR Green I. Laser-induced fluorescence detection is obtained by excitation at 488 nm and signal collection at 520 nm. Specificity and reproducibility of the protocols were established by using samples 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 number of hybridizations with radioactive probes, this present protocol is more rapid, giving sufficient separation on CZE after PCR reactions without preconcentration or desalting of samples.
Carrera
P
P
I.R.C.C.S., H.S. Raffaele, Milano, Italy.
Barbieri
A M
AM
Ferrari
M
M
Righetti
P G
PG
Perego
M
M
Gelfi
C
C
D000312:Adrenal Hyperplasia, Congenital; D000483:Alleles; D052160:Benzothiazoles; D003959:Diamines; D019075:Electrophoresis, Capillary; D005260:Female; D005456:Fluorescent Dyes; D005784:Gene Amplification; D006801:Humans; D007834:Lasers; D008297:Male; D009930:Organic Chemicals; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011804:Quinolines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013255:Steroid 21-Hydroxylase / Q000235:genetics*
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D013255:Steroid 21-Hydroxylase