Quikgene: A gene synthesis method integrated with ligation-free cloning
Quikgene: A gene synthesis method integrated with ligation-free cloning
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Quikgene：一种与无连接克隆集成的基因合成方法
DOI：
10.1016/j.ab.2011.04.004
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发表时间：
2011-08-01
影响因子：
2.9
通讯作者：
Han, Aidong
中科院分区：
文献类型：
--
作者：
Mao, Yanjun;Lin, Juanyu;Han, Aidong
基因合成是一种方便的工具，被广泛用于制造用于各种目的的基因。目前所有的方案基本上都采用由内而外的方法，使用DNA寡核苷酸或中间片段组装完整的基因。在这里，我们提出了一个有效的方法，集成基因合成和克隆到一个步骤。我们的方法是从QuikChange诱变发展而来的，可以修饰、延伸甚至从头合成相对较大的基因。将基因直接插入载体中，无需连接或亚克隆。我们从头合成了一个600 bp的基因，通过多个步骤的聚合酶链反应（PCR）直接进入细菌表达载体。这种由外向内的基因合成方法被称为Quikgene。此外，我们已经在插入引物中定义了至少9个核苷酸的重叠区域，其足以使PCR产物环化以进行有效转化，从而允许显著减少引物的长度。总之，我们的协议大大扩展了QuikChange插入的当前长度限制。更重要的是，它将基因合成和克隆合二为一。它在高通量结构基因组学方面具有潜在的应用价值。(C)2011 Elsevier Inc. All rights reserved.
Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or even de nova synthesize relatively large genes. The genes are inserted directly into vectors without ligations or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics. (C) 2011 Elsevier Inc. All rights reserved.