Addition of mirror-image L-DNA elements to DNA amplification circuits to distinguish leakage from target signal.
Addition of mirror-image L-DNA elements to DNA amplification circuits to distinguish leakage from target signal.
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DOI：
10.1016/j.bios.2021.113354
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发表时间：
2021-09-15
影响因子：
12.6
通讯作者：
Haselton FR
中科院分区：
文献类型：
--
作者：
Zimmers ZA;Adams NM;Haselton FR
依赖于由目标核酸触发的热力学驱动的杂交事件的DNA放大电路因其相对简单而被越来越多地使用。这些电路的一个缺点是，必须使用单独的“非靶标”控制反应来估计非特异性放大或电路泄漏，以消除假阳性。除了需要额外的反应外，这种方法的问题是很难为生物样本建立非靶标对照。为了克服这一局限性，我们提出了一种策略，将两种反应结合到同一管中，使用自然产生的右手D-DNA电路元件用于目标检测反应，使用相同的合成镜像左手L-DNA电路元件用于非靶标控制反应。我们使用催化发夹组装(CHA)来说明这种方法，这是研究最多的DNA扩增电路之一。在双手性ChA设计中，右手电路信号是由目标特定放大和电路漏电产生的，而左手电路信号是由电路漏电产生的。靶标特异性扩增是根据两个信号之间的差值计算的。这种双手性CHA反应的检测限与传统CHA反应的检测限相似(分别为81pM和92pM)。此外，在包括背景DNA、盐浓度增加、温度升高和尿液在内的广泛样本条件下，左手非靶标信号与右手泄漏信号相匹配。这些结果证明了双手性设计的稳健性和左手DNA在开发更适合于生物样品中的目标检测应用的新的DNA放大电路方面的潜在用途。
DNA amplification circuits that rely on thermodynamically-driven hybridization events triggered by a target nucleic acid are becoming increasingly utilized due to their relative simplicity. A drawback of these circuits is that non-specific amplification, or circuit leakage, must be estimated using a separate “no-target” control reaction to eliminate false positives. Aside from requiring an additional reaction, the problem with this approach is the difficulty of creating a no-target control for biological specimens. To overcome this limitation, we propose a strategy that combines both reactions into the same tube using naturally-occurring right-handed D-DNA circuit elements for the target detection reaction and identical synthetic mirror-image left-handed L-DNA circuit elements for the no-target control reaction. We illustrate this approach using catalyzed hairpin assembly (CHA), one of the most studied DNA amplification circuits. In a dual-chirality CHA design, the right-handed circuit signal is produced by target-specific amplification and circuit leakage, whereas the left-handed circuit signal is produced only by circuit leakage. The target-specific amplification is calculated as the difference between the two signals. The limit of detection of this dual-chirality CHA reaction was found to be similar to that of traditional CHA (81 vs 92 pM, respectively). Furthermore, the left-handed no-target signal matched the right-handed leakage across a wide range of sample conditions including background DNA, increased salt concentration, increased temperature, and urine. These results demonstrate the robustness of a dual-chirality design and the potential utility of left-handed DNA in the development of new DNA amplification circuits better-suited for target detection applications in biological samples.
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影响因子：
14.9
作者：
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DOI：
10.1073/pnas.0407024101
发表时间：
2004-10-26
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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