Highly specific unnatural base pair systems as a third base pair for PCR amplification.
Highly specific unnatural base pair systems as a third base pair for PCR amplification.
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DOI：
10.1093/nar/gkr1068
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发表时间：
2012-03
影响因子：
14.9
通讯作者：
Hirao I
中科院分区：
文献类型：
--
作者：
Yamashige R;Kimoto M;Takezawa Y;Sato A;Mitsui T;Yokoyama S;Hirao I
为了扩大DNA的遗传字母表，我们提出了高效的非天然碱基对系统作为PCR的人工第三碱基对。通过使用不同的聚合酶和模板序列环境以及修饰的Px碱基评估扩增效率和保真度，对7-（2-噻吩基）咪唑并[4，5-B]吡啶（Ds）和2-硝基-4-丙炔基吡咯（Px）之间的疏水非天然碱基对系统进行微调以用于有效的PCR。然后，我们发现Px碱基的一些修饰降低了模板中与天然碱基相对的非天然碱基底物的错误掺入率，而不降低Ds-Px配对选择性。在使用Deep Vent DNA聚合酶的优化条件下，错误掺入率极低（0.005%/bp/复制），这接近于聚合酶的天然碱基错配率。通过40个循环的PCR，具有不同序列背景的DNA片段被扩增超过1010倍，并且除了对不利的嘌呤-Ds-嘌呤基序的99.77%/复制之外，Ds-Px配对的选择性>99.9%/复制。此外，在100个循环（10个循环重复10次）后，DNA中>97%的Ds-Px对在1028倍扩增产物中存活。这种高度特异性的Ds-Px对系统为新的生物技术提供了一个框架。
Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds–Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼1010-fold by 40 cycles of PCR, and the selectivity of the Ds–Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds–Px pair in DNA survived in the 1028-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds–Px pair system provides a framework for new biotechnology.
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