Helicase-dependent isothermal DNA amplification
Helicase-dependent isothermal DNA amplification
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DOI：
10.1038/sj.embor.7400200
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发表时间：
2004-08-01
期刊：
影响因子：
7.7
通讯作者：
Kong, HM
中科院分区：
文献类型：
--
作者：
Vincent, M;Xu, Y;Kong, HM
聚合酶链式反应是目前应用最广泛的DNA体外扩增方法。然而，它需要热循环来分离两条DNA链。在体内，DNA通过带有各种辅助蛋白的DNA聚合酶进行复制，其中包括一种用于分离双链DNA的DNA解旋酶。我们模仿体内的这种机制，设计了一种新的体外等温DNA扩增方法。解旋酶依赖扩增(HDA)利用DNA解旋酶产生单链模板，用于引物杂交和随后的DNA聚合酶延伸。HDA不需要热循环。此外，与其他等温DNA扩增方法相比，它具有几个优点，反应流程简单，是真正的等温反应，可以在一个温度下进行整个过程。这些特性为开发简单的便携式DNA诊断设备提供了巨大的潜力，这些设备将在现场和护理点使用。
Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.