Litos Ioannis K, Ioannou Penelope C, Christopoulos Theodore K, Traeger-Synodinos Joanne, Kanavakis Emmanuel
Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece.
Anal Chem. 2007 Jan 15;79(2):395-402. doi: 10.1021/ac061729e.
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.
引物延伸（PEXT）反应是单核苷酸多态性（SNP）基因分型中应用最广泛的方法。目前分析PEXT反应产物的方法基于电泳、荧光共振能量转移、荧光偏振、焦磷酸测序、质谱、微阵列和光谱编码微球。我们报道了第一种无需仪器即可快速目视检测PEXT产物的干试剂试纸条方法。该方法应用于甘露糖结合凝集素基因（MBL2）中六个SNP的分析。通过PCR扩增跨越每个感兴趣SNP的基因组区域。使用等位基因特异性引物（针对一种或另一种变异核苷酸）进行两个引物延伸反应，这些引物在5'端含有寡聚（dA）片段。生物素-dUTP掺入延伸链中。将产物应用于试纸条，然后浸入适当的缓冲液中。当DNA通过毛细作用沿着试纸条移动时，它与寡聚（dT）功能化的金纳米颗粒杂交，使得只有延伸产物在测试区被固定的链霉亲和素捕获，产生一条红线。纳米颗粒与固定的寡聚（dA）杂交在试纸条的对照区形成第二条红线。试纸条检测在10分钟内完成。我们使用来自27名患者的基因组DNA分析了甘露糖结合凝集素基因（MBL2）的六个SNP，总共代表74个变异核苷酸位置。患者基因型与直接DNA测序数据显示100%一致。所描述的PEXT-试纸条检测快速且高度准确；它不需要专门的仪器或训练有素的技术人员。它适用于每个患者检测少数选定SNP标记且每次检测成本较低的诊断实验室。
