Department of Chemistry, Capital Normal University, Beijing, China.
ACS Appl Mater Interfaces. 2013 Jul 10;5(13):6276-84. doi: 10.1021/am4013209. Epub 2013 Jun 28.
Recently, the applications of nanomaterial-assisted polymerase chain reaction (nanoPCR) have received considerable attention. Several potential mechanisms have been proposed, but mainly according to the results of PCR assays under specific conditions and lacking direct and general evidence. The mechanism of nanoPCR has not been elucidated yet. Here, taking gold nanoparticles (AuNPs) as an example, we report the three general effects of AuNPs: (1) AuNPs adsorb polymerase and modulate the amount of active polymerase in PCR, which was directly demonstrated by a simple and straightforward colorimetric assay and the dynamic light scattering measurements. (2) AuNPs adsorb primers and decrease the melting temperatures (Tm) of the duplexes formed with perfectly matched and mismatched primers and increase the Tm difference between them. (3) AuNPs adsorb PCR products and facilitate the dissociation of them in the denaturing step. All these effects were confirmed by addition of a rationally selected surface adsorbent, bovine thrombin, to highly efficiently modulate the surface adsorption of PCR components. These findings suggested that AuNPs should have multiple effects on PCR: (1) to regulate PCR in a case-by-case way via modulating the amount of active polymerase in PCR; (2) to improve PCR specificity in the annealing step via increasing the Tm difference between the perfectly matched and mismatched primers; (3) to improve PCR efficiency via speeding up the dissociation of the PCR products in the denaturing step. Taken together, we proposed the mechanism of nanoPCR is that the surface interaction of PCR components (polymerase, primers, and products) with AuNPs regulates nanoPCR. We further demonstrated that the applications of these findings improve the PCR of the amelogenin genes and Hepatitis B virus gene for genetic analysis. These findings could also provide helpful insight for the applications of other nanomaterials in nanoPCR.
最近，纳米材料辅助聚合酶链反应（nanoPCR）的应用受到了相当多的关注。已经提出了几种潜在的机制，但主要是根据特定条件下的 PCR 分析结果得出的，缺乏直接和普遍的证据。nanoPCR 的机制尚未阐明。在这里，我们以金纳米粒子（AuNPs）为例，报道了 AuNPs 的三个一般效应：（1）AuNPs 吸附聚合酶并调节 PCR 中活性聚合酶的数量，这通过简单直接的比色测定法和动态光散射测量直接证明。（2）AuNPs 吸附引物并降低与完全匹配和不匹配引物形成的双链体的熔点（Tm），并增加它们之间的 Tm 差异。（3）AuNPs 吸附 PCR 产物并促进它们在变性步骤中解离。所有这些效应都通过添加合理选择的表面吸附剂牛凝血酶来证实，这可以高效地调节 PCR 成分的表面吸附。这些发现表明 AuNPs 对 PCR 应该有多种影响：（1）通过调节 PCR 中活性聚合酶的数量来有针对性地调节 PCR；（2）通过增加完全匹配和不匹配引物之间的 Tm 差异来提高 PCR 特异性；（3）通过加速变性步骤中 PCR 产物的解离来提高 PCR 效率。综上所述，我们提出 nanoPCR 的机制是 PCR 组件（聚合酶、引物和产物）与 AuNPs 的表面相互作用调节 nanoPCR。我们进一步证明，这些发现的应用可以改善遗传分析中 amelogenin 基因和乙型肝炎病毒基因的 PCR。这些发现还可以为其他纳米材料在 nanoPCR 中的应用提供有价值的见解。
