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  • 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.
    Author: Brugère JF, Gobron S, Baud E, Cailloux F.
    Journal: Mol Cell Probes; 2008; 22(5-6):320-3. PubMed ID: 18657606.
    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.
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