Design and validation of a colorimetric test for the genetic diagnosis of hemochromatosis using α-phosphorothioate nucleotides.

Hereditary hemochromatosis is an autosomal recessive disease highly prevalent in Northern Europe. Here we describe the performance of a genetic test for two mutations of the HFE gene (C282Y and H63D). It is based on a solid-phase PCR coupled with an α-phosphorothioate-mediated primer extension, conferring resistance to hydrolysis by ExoIII.

Single-tube genotyping using a solid-phase method that combines alpha-phosphorothioate-mediated primer extension and ExoIII: proof of concept with the F508del cystic fibrosis diagnosis.

Detection of single nucleotide polymorphisms (SNPs) and of mutations is of importance in the field of genetics, biomedical research and in vitro diagnosis. We report here a genotyping procedure that can be virtually applied to any locus within a genome: it uses alpha-phosphorothioate deoxynucleotides in a primer-extension step followed by an ExoIII treatment. Non-extended primers are hydrolyzed whereas extended primers resist this treatment, indicating which nucleotide has been incorporated, i.