Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix

Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the mos t common mutation in the 21-hydroxylase gene is presented using primer exte nsion and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DN A samples were subjected to polymerase chain reaction (PCR) in order to amp lify a 422 bp fragment of the CYP21 gene containing the single nucleotide p olymorphism (SNP) site. This product served as a template in the primer ext ension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis usin g 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labe led primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.