The primer extension (PEXT) reaction is the most widely used approach to genotyping of single-nucleotide polymorphisms (SNPs). Current methods for analysis of PEXT reaction products are based on electrophoresis, fluorescence resonance energy transfer, fluorescence polarization, pyrosequencing, mass spectrometry, microarrays, and spectrally encoded microspheres. We report the first dry-reagent dipstick method that enables rapid visual detection of PEXT products without instrumentation. The method is applied to the analysis of six SNPs in the mannose-binding lectin gene (MBL2). The genomic region that spans each SNP of interest is amplified by PCR. Two primer extension reactions are performed with allele-specific primers (for one or the other variant nucleotide), which contain an oligo(dA) segment at the 5'-end. Biotin-dUTP is incorporated in the extended strand. The product is applied to the strip followed by immersion in the appropriate buffer. As the DNA moves along the strip by capillary action, it hybridizes with oligo(dT)-functionalized gold nanoparticles, such that only extended products are captured by immobilized streptavidin at the test zone, generating a red line. A second red line is formed at the control zone of the strip by hybridization of the nanoparticles with immobilized oligo(dA). The dipstick test is complete within 10 min. We analyzed six SNPs of the mannose-binding lectin gene (MBL2) using genomic DNA from 27 patients, representing a total of 74 variant nucleotide positions. Patient genotypes showed 100% concordance with direct DNA sequencing data. The described PEXT-dipstick assay is rapid and highly accurate; it does not require specialized instrumentation or highly trained technical personnel. It is appropriate for a diagnostic laboratory where a few selected SNP markers are examined per patient with a low cost per assay.

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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.