Abstrakt
Objective: The amplification-created restriction site method was performed to establish laboratory procedure for routine molecular genetic analysis of steroid 21-hydroxylase gene. Subjects and Methods: Patients and healthy controls (homozygotes and heterozygotes) were analysed for eight common CYP21 gene mutations (P30L, A/C655G, 8bp deletion in exon, 3, I172N, V281L, Q318X, R356W and P453S) by modification of the amplification-created restriction site method.
CYP21 gene was amplified with gene specific primers. Nested polymerase chain reactions were performed using mutagenic primers to introduce restriction sites as needed.
Digestion by restriction endonucleases of the polymerase chain reaction products was used to detect the presence of mutations. Results: The presented modification of amplification-created restriction site method is more rapid and efficient, shows improved sensitivity, specificity, and ability to distinguish allelic heterozygosity.
Conclusion: The presented modification of amplification-created restriction site inexpensive method is suitable for a routine CYP21 gene mutation analysis.