Vincent Myriam, Xu Yan, Kong Huimin
New England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, USA.
EMBO Rep. 2004 Aug;5(8):795-800. doi: 10.1038/sj.embor.7400200. Epub 2004 Jul 9.
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.
聚合酶链反应是体外DNA扩增最广泛使用的方法。然而,它需要热循环来分离两条DNA链。在体内,DNA由DNA聚合酶与各种辅助蛋白一起复制,其中包括一种用于分离双链DNA的DNA解旋酶。我们通过模拟这种体内机制设计了一种新的体外等温DNA扩增方法。解旋酶依赖性扩增(HDA)利用DNA解旋酶生成单链模板,用于引物杂交以及随后由DNA聚合酶进行的引物延伸。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,反应方案简单,是一种真正的等温反应,整个过程可在一个温度下进行。这些特性为开发用于现场和即时护理的简单便携式DNA诊断设备提供了巨大潜力。