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Rapid genotyping of CYP2D6, CYP2C19 and TPMT polymorphisms by primer extension reaction in a dipstick format

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Abstract

In recent years an increasing amount of interest has been directed at the study and routine testing of polymorphisms responsible for variations in drug metabolism. Most of the current methods involve either time-consuming electrophoresis steps or specialized and expensive equipment. In this context, we have developed a rapid, simple and robust method for genotyping of CYP2D6*3, CYP2D6*4, CYP2C19*2, CYP2C19*3 and TPMT*2 single nucleotide polymorphisms (SNP). Genomic DNA is isolated from whole blood and the segments that span the SNP of interest are amplified by PCR. The products are subjected directly (without purification) to two primer extension (PEXT) reactions (three cycles each) using normal and mutant primers in the presence of biotin-dUTP. The PEXT primers contain a (dA)30 segment at the 5′ end. The PEXT products are detected visually by a dry-reagent dipstick-type assay in which the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functionalized gold nanoparticles. Patient samples (76 variants in total) were genotyped and the results were fully concordant with those obtained by direct DNA sequencing.

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Acknowledgements

This work was supported by a University–Industry collaboration grant (EPAN) funded by the General Secretariat of Research and Technology (GSRT) of Greece and by Medicon Hellas S.A. (Gerakas, Greece 15344).

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Author notes

  1. Evaggelia Emmanouilidou

    Present address: Division of Basic Neurosciences, Foundation for Biomedical Research of the Academy of Athens, Soranou Efesiou 4, 11527, Athens, Greece

Authors and Affiliations

  1. Department of Chemistry, University of Athens, 15771, Athens, Greece

    Ioannis K. Litos, Evaggelia Emmanouilidou & Penelope C. Ioannou

  2. Medicon Hellas SA, 15344, Gerakas, Greece

    Kyriaki M. Glynou

  3. Unit of Metabolic Diseases, Choremio Research Laboratory, 1st Department of Pediatrics, “Aghia Sophia” Children’s Hospital, University of Athens, 11527, Athens, Greece

    Eleftheria Laios

  4. Department of Chemistry, University of Patras, 26500, Patras, Greece

    Theodore K. Christopoulos

  5. Foundation for Research and Technology Hellas- Institute of Chemical Engineering and High Temperature Chemical Processes, P.O. Box 1414, 26500, Patras, Greece

    Theodore K. Christopoulos

  6. School of Medicine, University of Crete, 71110, Heraklion, Greece

    Marilena Kampa, Elias Castanas & Achille Gravanis

Authors
  1. Ioannis K. Litos
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  2. Evaggelia Emmanouilidou
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  3. Kyriaki M. Glynou
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  4. Eleftheria Laios
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  5. Penelope C. Ioannou
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  6. Theodore K. Christopoulos
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  7. Marilena Kampa
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  8. Elias Castanas
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Corresponding author

Correspondence to Penelope C. Ioannou.

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Litos, I.K., Emmanouilidou, E., Glynou, K.M. et al. Rapid genotyping of CYP2D6, CYP2C19 and TPMT polymorphisms by primer extension reaction in a dipstick format. Anal Bioanal Chem 389, 1849–1857 (2007). https://doi.org/10.1007/s00216-007-1593-4

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  • DOI: https://doi.org/10.1007/s00216-007-1593-4

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