Enzymatic assembly of DNA molecules up to several hundred kilobases
Enzymatic assembly of DNA molecules up to several hundred kilobases
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DOI：
10.1038/nmeth.1318
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发表时间：
2009-05-01
期刊：
影响因子：
48
通讯作者：
Smith, Hamilton O.
中科院分区：
文献类型：
--
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
我们描述了一个等温的，单一的反应方法组装多个重叠的DNA分子的5'核酸外切酶，DNA聚合酶和DNA连接酶的协同作用。首先，我们凹陷DNA片段，产生单链DNA突出端，特异性退火，然后共价连接它们。这种组装方法可用于无缝构建合成和天然基因、遗传途径和整个基因组，并且可能是有用的分子工程工具。
We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.