Rapid genotyping of CYP2D6, CYP2C19 and TPMT polymorphisms by primer extension reaction in a dipstick format.
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.
Litos
Ioannis K
IK
Department of Chemistry, University of Athens, 15771 Athens, Greece.
Emmanouilidou
Evaggelia
E
Glynou
Kyriaki M
KM
Laios
Eleftheria
E
Ioannou
Penelope C
PC
Christopoulos
Theodore K
TK
Kampa
Marilena
M
Castanas
Elias
E
Gravanis
Achille
A
D020121:5' Untranslated Regions; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D065731:Cytochrome P-450 CYP2C19; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D016678:Genome; D005838:Genotype; D008780:Methyltransferases / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
D020121:5' Untranslated Regions; D017931:DNA Primers; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D001710:Biotin; D019809:Streptavidin; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D019389:Cytochrome P-450 CYP2D6; D008780:Methyltransferases; C022745:thiopurine methyltransferase