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.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.
Brugère
Jean-François
JF
Nucleica S.A., Biopole Clermont-Limagne, Saint-Beauzire, France. J-Francois.BRUGERE@u-clermont1.fr
Gobron
Stéphane
S
Baud
Eric
E
Cailloux
Fabrice
F
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*
D017931:DNA Primers; D010710:Phosphates; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C035638:thiophosphoric acid