title: 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.
abstract: 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.e. the genotype of the locus. A 3-bp deletion in the CFTR gene (F508del, the most prevalent mutation involved in cystic fibrosis) was used as a model, in a single-tube procedure for each nucleotide to be tested. Human genomic DNA samples were correctly genotyped in less than 3h by a solid-phase PCR followed by primer extension, ExoIII treatment and an ELISA-like detection method. The same principle (primer extension with alpha-phosphorothioate deoxynucleotide, ExoIII treatment) should also be combined with other detection systems such as gel or capillary electrophoresis, mass spectrometry or DNA chips.
authors.affiliation: Nucleica S.A., Biopole Clermont-Limagne, Saint-Beauzire, France. J-Francois.BRUGERE@u-clermont1.fr
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Brugère
authors.forename: Stéphane
authors.initials: S
authors.lastname: Gobron
authors.forename: Eric
authors.initials: E
authors.lastname: Baud
authors.forename: Fabrice
authors.initials: F
authors.lastname: Cailloux
mesh_terms: D001483:Base Sequence; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
chemical_list: D017931:DNA Primers; D010710:Phosphates; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C035638:thiophosphoric acid