Toubanaki Dimitra K, Christopoulos Theodore K, Ioannou Penelope C, Gravanis Achille
Department of Chemistry, University of Patras, Rio Patras, Greece GR-26500.
Pharmacogenomics. 2009 Mar;10(3):495-504. doi: 10.2217/14622416.10.3.495.
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 CYP2C192 (c.681G>A) and CYP2D64 (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.
We genotyped 55 samples for CYP2C192 and 49 samples for CYP2D64. The accuracy of this method was confirmed by sequencing and electrophoresis.
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.
单核苷酸多态性（SNP）分型策略包括指数扩增步骤、等位基因鉴别反应及产物检测。通常，等位基因鉴别在扩增后进行。四引物聚合酶链反应（PCR）可在扩增步骤中实现等位基因鉴别，从而避免额外的基因分型反应。然而，迄今为止，电泳是检测四引物PCR产物的唯一方法。我们报告了一种试纸条检测法，可在数分钟内无需仪器实现对四引物PCR产物的可视化检测。该方法应用于CYP2C192（c.681G>A）和CYP2D64（g.3465G>A）的基因分型。
一对外部引物扩增包含SNP的片段。生物素化的内部引物有3个错配，并与外部引物配对以引导双向扩增，产生等位基因特异性片段。产物与聚（dA）尾探针短暂杂交，然后应用于DNA生物传感器，再将其浸入适当的缓冲液中。随着缓冲液沿生物传感器迁移，杂交体在检测区被链霉亲和素捕获，并与寡聚（dT）功能化的金纳米颗粒相互作用，导致形成一条红线。在对照区形成另一条红线，以表明传感器功能正常。
我们对55个样本进行了CYP2C192基因分型，对49个样本进行了CYP2D64基因分型。通过测序和电泳证实了该方法的准确性。
所提出方法的独特优势在于其简单性和低成本。与电泳不同，杂交可提供扩增片段的序列确认。干试剂试纸条形式最大限度地减少了对高素质人员的需求。
