The development and characterization of a new enzyme reaction contribute to advancements in modern biotechnology. Here, we report a novel CIS (clamping-mediated incorporation of single-stranded DNA with concomitant DNA synthesis) reaction catalyzed by Taq polymerase. In the reaction, a single-stranded DNA (ssDNA) with 3′ Cs is attached with a preformed 3′ G-tail of double-stranded DNA (dsDNA); DNA syntheses starting from both 3′ ends result in the incorporation of ssDNA. A 3′ G-tail length of 3 nucleotides adequately supports this reaction, indicating that Taq polymerase can clump short Watson–Crick base pairs as short as three pairs and use them to initiate DNA polymerization. The reverse transcriptase from Molony murine leukemia virus catalyzes strand displacement synthesis and produces flapped-end DNA, whereas the reaction by Taq polymerase involves the nick translation. These new reaction properties may be beneficial for the development of new molecular tools applicable in various fields. Apart from its CIS reaction activity, we also report that Taq polymerase has the undesirable characteristic of removing 5' fluorescent labels from dsDNA. This characteristic may have compromised various experiments involving the preparation of fluorescently-labeled dsDNA by PCR for a long time.

新酶反应的开发和表征有助于现代生物技术的进步。在这里，我们报道了一种由 Taq 聚合酶催化的新型 CIS （clamping 介导的单链 DNA 与伴随的 DNA s结合）反应。在反应中，具有 3' Cs 的单链 DNA （ssDNA） 与双链 DNA （dsDNA） 的预制 3' G 尾相连;从两个 3' 末端开始的 DNA 合成导致 ssDNA 的掺入。3 个核苷酸的 3′ G 尾长度足以支持该反应，表明 Taq 聚合酶可以聚集短至 3 对的短 Watson-Crick 碱基对，并使用它们来引发 DNA 聚合。来自 Molony 鼠白血病病毒的逆转录酶催化链置换合成并产生瓣状末端 DNA，而 Taq 聚合酶的反应涉及切口翻译。这些新的反应性质可能有利于开发适用于各个领域的新分子工具。除了 CIS 反应活性外，我们还报道了 Taq 聚合酶具有从 dsDNA 中去除 5' 荧光标记的不良特性。这一特性可能已经影响了很长时间内涉及通过 PCR 制备荧光标记 dsDNA 的各种实验。