Helicase-dependent amplification of nucleic acids.
Helicase-dependent amplification of nucleic acids.
复制标题
DOI：
10.1002/0471142727.mb1511s104
复制
发表时间：
2013-10-11
影响因子：
--
通讯作者：
Lemieux, Bertrand
中科院分区：
文献类型：
--
作者：
Cao, Yun;Kim, Hyun-Jin;Lemieux, Bertrand
解旋酶依赖性扩增（HDA）是一种新型的核酸等温体外扩增方法。HDA反应通过延伸两个寡核苷酸引物选择性扩增靶序列。与聚合酶链式反应（PCR）不同，HDA使用解旋酶来分离脱氧核糖核酸（DNA）链，而不是热变性。这允许DNA扩增而不需要热循环。在HDA中使用的解旋酶是从嗜热生物腾冲嗜热厌氧微生物（Thermoanaerobactertengcongensis，TteUvrD）获得的解旋酶超家族II蛋白。这种热稳定解旋酶能够在升高的温度（60° C至65°C）下解旋平端核酸底物。HDA反应还可以与用于核糖核酸（RNA）扩增的逆转录偶联。当在荧光计中进行孵育时，可以在反应期间使用荧光探针检测该反应的产物。或者，可以在扩增后使用含有嵌入的侧向流动条的一次性扩增子容纳装置检测产物。
Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip.