Kerman Kagan, Vestergaard Mun'delanji, Nagatani Naoki, Takamura Yuzuru, Tamiya Eiichi
School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
Anal Chem. 2006 Apr 1;78(7):2182-9. doi: 10.1021/ac051526a.
The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The attachment of long single-strands on GCE surface caused the accumulation of [Co(NH3)6]3+ and a high current response. Here, we report a versatile method that would allow for simple and rapid analysis of nucleic acids in combination with PNA-mediated PCR and asymmetric PCR techniques by using an electrochemical genosensor.
肽核酸（PNA）具有独特的结构，它连接着N-（2-氨基乙基）甘氨酸单元，形成一个中性骨架，并阻止其作为DNA聚合酶的引物。这种结构已被用于一种电化学生物传感器方案中，用于简单而灵敏地检测杂交。当PNA靶向基因上的单核苷酸多态性（SNP）或野生型位点时，PNA介导的聚合酶链反应（PCR）钳夹方法可有效阻断PCR产物的形成。在我们的报告中，用于PCR钳夹的PNA探针靶向乙醇脱氢酶的野生型位点。使用差分脉冲伏安法监测带负电荷的DNA与带有氧化还原活性金属阳离子六氨合钴（III）（[Co(NH3)6]3+）的中性PNA分子之间的静电相互作用。[Co(NH3)6]3+与DNA的静电结合为区分PNA/PNA、PNA/DNA和DNA/DNA杂交分子提供了基础。我们优化了实验条件，如探针浓度、[Co(NH3)6]3+浓度、[Co(NH3)6]3+的积累时间和靶标浓度。还采用了一种新的预处理方法，以便在玻碳电极（GCE）表面快速简单地检测PCR扩增子与探针之间的杂交反应。该方法基于高温处理（95℃，5分钟），然后在DNA引物存在下孵育1分钟。过量的DNA引物可防止变性链重新杂交，同时使靶基因序列与固定化探针结合。此外，采用不对称PCR检测标准抗草甘膦转基因大豆样品中是否存在转基因生物。由于引物浓度不均，不对称PCR的扩增子主要是单链DNA，它能与传感器表面的DNA探针有效杂交。长单链附着在GCE表面会导致[Co(NH3)6]3+积累并产生高电流响应。在此，我们报告一种通用方法，该方法可通过使用电化学生物传感器，结合PNA介导的PCR和不对称PCR技术，对核酸进行简单快速的分析。
