Polymerase Chain Reaction using "V" Shape Thermal Cycling Program
Polymerase Chain Reaction using "V" Shape Thermal Cycling Program
复制标题
使用“V”形热循环程序的聚合酶链式反应
DOI：
10.7150/thno.31986
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发表时间：
2019-01-01
期刊：
影响因子：
12.4
通讯作者：
Tang, Zhuo
中科院分区：
文献类型：
--
作者：
Chen, Rong;Lu, Xue;Tang, Zhuo
聚合酶链反应（PCR）是分子生物学和诊断学中最常用的技术。为了实现更快的PCR反应时间，之前的研究采用了两种策略。这包括通过开发新的设备来提高热斜坡率，以减少浪费在温度转换上的时间，并缩短每一步的保持时间，这甚至可能导致放大效率的妥协。因此，需要进一步改进的technology.Methods：一种不同的方式来实现快速DNA扩增的开发，通过使用以前认为浪费的时间花在加热和冷却的样品，以完成扩增。结果：与常规PCR扩增方法相比，扩增时间可节省2/3。此外，通过使用具有更高解链温度（Tm）的更长引物可以进一步缩短反应时间。在普通PCR仪上进行的“V”型聚合酶链反应（VPCR）扩增98 bp片段的记录时间为8 min，且保留了传统PCR技术的特异性、敏感性、通用性和与定量检测兼容性等优点。证实了当循环机以中等的热斜坡速率运行时，PCR的三个程序可以在动态加热和冷却过程中完成。由于VPCR是在现有PCR系统的基础上发展起来的，因此它可以立即在任何生物实验室中实施，为从事生命科学和人类健康领域工作的人们提供了极大的方便。
Polymerase chain reaction (PCR) is the most commonly used technique in molecular biology and diagnostics. To achieve faster PCR reaction time, two strategies were employed by previous studies. That includes improving the thermal ramp rate by developing novel devices to reduce the time wasted on temperature transitions and cutting the holding time in every step, which could even lead to compromise in amplification efficiency. Hence the need to further improve the technique.Methods: A different way to achieve fast DNA amplification is developed by using the previously thought wasted time spent on heating and cooling the samples to finish the amplification. That means the holding time of the three procedures are omitted and this could be carried out on the ordinary PCR thermal cyclers.Results: 2/3 of the amplification time is easily saved, compared to the conventionally used method. Additionally, the reaction time could be further reduced by using longer primers with higher melting temperature (Tm). The record time of the "V" shape Polymerase chain reaction (VPCR) conducted on ordinary PCR machine for amplification of a 98 bp fragment is 8 min. Furthermore, VPCR still retains the merits of traditional PCR technique, including specificity, sensitivity, generality, and compatibility with quantitative detection.Conclusion: It is confirmed that the three procedures of PCR could be completed during the dynamic heating and cooling process when the cyclers are run at a moderate thermal ramp rate. As VPCR described here is based on the current PCR system, it could be implemented in any biological Lab immediately and provide great convenience to the people working in the field of life science and human health.