An efficient unnatural base pair for PCR amplification
An efficient unnatural base pair for PCR amplification
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DOI：
10.1021/ja073830m
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发表时间：
2007-12-19
影响因子：
15
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Hirao, Ichiro;Mitsui, Tsuneo;Yokoyama, Shigeyuki
通过非自然碱基对系统扩展遗传字母表为现代生物技术提供了强有力的工具。作为以前非天然碱基对的替代，我们开发了一种新的7-（2-噻吩基）咪唑并[4，5-B]吡啶（Ds）和2-硝基吡咯（Pn）之间的碱基对，其在DNA扩增中起作用。Pn在复制中比另一个先前报道的配对伙伴吡咯-2-甲醛（Pa）更选择性地与Ds配对。Pin的硝基有效地防止了与A的错配。以Ds的γ-氨基三磷酸盐和Pn的常用三磷酸盐及天然碱基为底物，以Vent DNA聚合酶为3'至5'核酸外切酶活性的聚合酶，实现了Ds-Pn对的高效率和高选择性PCR扩增。经过20个循环的PCR扩增后，扩增片段中Ds-Pn位点的总突变率接近1%。含有非天然Ds-Pn对的DNA片段的PCR扩增将用于基于DNA的生物技术中的扩展遗传系统。
Expansion of the genetic alphabet by an unnatural base pair system provides a powerful tool for modern biotechnology. As an alternative to previous unnatural base pairs, we have developed a new pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which functions in DNA amplification. Pn more selectively pairs with Ds in replication than another previously reported pairing partner, pyrrole-2-carbaldehyde (Pa). The nitro group of Pin efficiently prevented the mispairing with A. High efficiency and selectivity of the Ds-Pn pair in PCR amplification were achieved by using a substrate mixture of the gamma-amidotriphosphate of Ds and the usual triphosphates of Pn and the natural bases, with Vent DNA polymerase as a 3' to 5' exonuclease-proficient polymerase. After 20 cycles of PCR, the total mutation rate of the Ds-Pn site in an amplified DNA fragment was similar to 1%. PCR amplification of DNA fragments containing the unnatural Ds-Pn pair would be useful for expanded genetic systems in DNA-based biotechnology.