Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.
Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.
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DOI：
10.1093/nar/gkac1221
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发表时间：
2023-01-11
影响因子：
14.9
通讯作者：
Ellington, Andrew D.
中科院分区：
文献类型：
--
作者：
Dangerfield, Tyler L.;Paik, Inyup;Bhadra, Sanchita;Johnson, Kenneth A.;Ellington, Andrew D.
环介导的等温扩增（LAMP）已被证明比PCR更容易实现即时诊断测试。然而，LAMP的潜在机制是复杂的，并且LAMP中的主要步骤的动力学尚未完全阐明，这阻碍了检测开发的合理改进。在这里，我们提出了我们的工作，以表征在LAMP的基本步骤的动力学，并表明：（i）链侵入/引发是在LAMP反应的限速步骤;（ii）环引物在加速起始速率中起重要作用，并且在指数扩增阶段中不单独起作用，以及（iii）通过Bst-LF聚合酶的链置换合成相对快（125 nt/s）和进行性，尽管在高GC含量模板上具有一些中断。在这些数据的基础上，我们能够开发出一个动力学模型，该模型将各个动力学实验与本体LAMP反应联系起来。这里开发的检测方法为LAMP的机制提供了重要的见解，整个模型在设计更灵敏和更快的LAMP反应中应该是至关重要的。我们采用的动力学方法可能证明与其他等温DNA扩增方法有用。
Loop-mediated isothermal amplification (LAMP) has proven to be easier to implement than PCR for point-of-care diagnostic tests. However, the underlying mechanism of LAMP is complicated and the kinetics of the major steps in LAMP have not been fully elucidated, which prevents rational improvements in assay development. Here we present our work to characterize the kinetics of the elementary steps in LAMP and show that: (i) strand invasion / initiation is the rate-limiting step in the LAMP reaction; (ii) the loop primer plays an important role in accelerating the rate of initiation and does not function solely during the exponential amplification phase and (iii) strand displacement synthesis by Bst-LF polymerase is relatively fast (125 nt/s) and processive on both linear and hairpin templates, although with some interruptions on high GC content templates. Building on these data, we were able to develop a kinetic model that relates the individual kinetic experiments to the bulk LAMP reaction. The assays developed here provide important insights into the mechanism of LAMP, and the overall model should be crucial in engineering more sensitive and faster LAMP reactions. The kinetic methods we employ should likely prove useful with other isothermal DNA amplification methods.
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DOI：
10.1016/j.jbc.2021.101451
发表时间：
2022-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Dangerfield TL;Kirmizialtin S;Johnson KA
通讯作者：
Johnson KA
影响因子：
4.7
作者：
Paik, Inyup;Bhadra, Sanchita;Ellington, Andrew D.
通讯作者：
Ellington, Andrew D.
影响因子：
2.9
作者：
Dangerfield TL;Huang NZ;Johnson KA
通讯作者：
Johnson KA
影响因子：
4.1
作者：
Krolov, Katrin;Frolova, Jekaterina;Langel, Olo, I
通讯作者：
Langel, Olo, I
影响因子：
7.4
作者：
Cai, Sheng;Jung, Cheulhee;Ellington, Andrew D.
通讯作者：
Ellington, Andrew D.