Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products.
Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products.
复制标题
多重PCR：使用热稳定的热嗜热RECA蛋白来最大程度地减少非特异性PCR产物。
DOI：
10.1093/nar/gni111
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发表时间：
2005-08-08
影响因子：
14.9
通讯作者：
Oishi, Michio
中科院分区：
文献类型：
--
作者：
Shigemori, Yasushi;Mikawa, Tsutomu;Shibata, Takehiko;Oishi, Michio
在本文中，我们报道了在PCR中包含来自嗜热细菌Thermus thermophilus的耐热RecA蛋白及其辅助因子（ATP），可以有效地消除非特异性PCR产物。RecA蛋白在基因重组中具有催化同源DNA分子配对的高保真度作用，其作用是由于其促进了PCR中的精确引物（即在引物序列与靶序列完全互补的位点上引物）。此外，RecA蛋白大大降低了PCR所需的引物浓度。这些实验结果导致多重PCR的实现，即在同一反应混合物中对多个位点进行PCR。我们能够在不影响PCR产物扩增模式的情况下，成功地进行十几次反应的多重PCR。
In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products.