Visual genotyping of SNPs of drug-metabolizing enzymes by tetra-primer PCR coupled with a dry-reagent DNA biosensor

摘要

Background: SNP-typing strategies involve an exponential amplification step, an allele discrimination reaction and detection of the products. Usually, allele discrimination is performed after amplification. Tetra-primer PCR allows allele discrimination during the amplification step, thereby avoiding additional genotyping reactions. However, to date, electrophoresis is the only method used for detection of tetra-primer PCR products. We report a dipstick test that enables visual detection of tetra-primer PCR products within minutes without instruments. The method is applied to the genotyping of CYP2C19*2 (c.681G>A) and CYP2D6*4 (g.3465G>A). Materials & methods: A pair of external primers amplifies a segment encompassing the SNPs. Biotinylated inner primers have a 3´-mismatch and pair off with the external primers to guide a bidirectional amplification that generates allele-specific fragments. The products are hybridized briefly with poly(dA)-tailed probes and applied to the DNA biosensor, which is then immersed in the appropriate buffer. As the buffer migrates along the biosensor, the hybrids are captured from streptavidin at the test zone and interact with oligo(dT)-functionalized gold nanoparticles leading to the formation of a red line. Another red line is formed at the control zone to indicate proper function of the sensor. Results: We genotyped 55 samples for CYP2C19*2 and 49 samples for CYP2D6*4. The accuracy of this method was confirmed by sequencing and electrophoresis. Conclusions: The unique advantages of the proposed method are its simplicity and low cost. Contrary to electrophoresis, hybridization provides sequence confirmation of amplified fragments. The dry-reagent dipstick format minimizes the requirements for highly qualified personnel.