The role of reactivity in DNA templated native chemical PNA ligation during PCR.
The role of reactivity in DNA templated native chemical PNA ligation during PCR.
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PCR 过程中 DNA 模板天然化学 PNA 连接反应活性的作用
DOI：
10.1016/j.bmc.2013.04.064
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发表时间：
2013
影响因子：
3.5
通讯作者：
O. Seitz
中科院分区：
文献类型：
--
作者：
A. Roloff;O. Seitz
DNA模板化的荧光反应已被用作核酸序列特异性检测的诊断工具;并且已显示硫酯-和1，2-氨基硫醇-修饰的PNA探针之间的天然化学连接是报道的最具选择性的DNA检测方法之一。我们探讨了DNA模板化反应是否可以与聚合酶链反应（PCR）相结合。这一奋进构成了一个重大挑战。所涉及的反应基团必须足够稳定以耐受PCR过程中施加的高温。然而，连接反应必须非常快速地进行，并且在退火和引物延伸步骤中可用的短时间内特异性测序，然后在大约10分钟后使用变性。1分钟，开始下一个PCR循环。这需要仔细优化的三元复合物架构，以及探针长度和探针反应性的调整。我们的研究结果指出了连接结构的首要性。我们表明，一旦合适的退火位点已被确定，反应性较低的探针集可能是优选的，如果序列特异性是主要关注。反应性调节使得能够开发PCR内连接，其用于人BRaf基因中V600 E（T1799 A）点突变的单核苷酸特异性分型。显示了天然化学PNA连接的效率和序列特异性，阿托摩尔模板证明足以触发信号，而1000倍更高负载的单个错配模板未能诱导可感知的信号。
DNA templated fluorogenic reactions have been used as a diagnostic tool for the sequence specific detection of nucleic acids; and it has been shown that the native chemical ligation between thioester- and 1,2-aminothiol-modified PNA probes is amongst the most selective DNA detection methods reported. We explored whether a DNA templated reaction can be interfaced with the polymerase chain reaction (PCR). This endeavor posed a significant challenge. The reactive groups involved must be sufficiently stable to tolerate the high temperature applied in the PCR process. Nevertheless, the ligation reaction must proceed very rapidly and sequence specifically within the short time available in the annealing and primer extension steps before denaturation is used after approx. 1min to commence the next PCR cycle. This required a careful optimization of the ternary complex architecture as well as adjustments of probe length and probe reactivities. Our results point to the prime importance of the ligation architecture. We show that once suitable annealing sites have been identified less reactive probe sets may be preferable if sequence specificity is of major concern. The reactivity tuning enabled the development of an in-PCR ligation, which was used for the single nucleotide specific typing of the V600E (T1799A) point mutation in the human BRaf gene. Showcasing the efficiency and sequence specificity of native chemical PNA ligation, attomolar template proofed sufficient to trigger signal while a 1000-fold higher load of single mismatched template failed to induce appreciable signal.
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