A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences
A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences
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DOI：
10.1093/nar/gnh094
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发表时间：
2004-07-01
影响因子：
14.9
通讯作者：
Li, Y
中科院分区：
文献类型：
--
作者：
Xiong, AS;Yao, QH;Li, Y
DNA 序列的化学合成为修饰基因和研究基因功能、结构和表达提供了强大的工具。在这里，我们报告了一种简单、高保真且经济高效的基于 PCR 的两步 DNA 合成 (PTDS) 方法，用于合成长 DNA 片段。该方法涉及两个步骤。 (i) 感兴趣DNA的单个片段的合成：将10至12个具有20 bp重叠的60mer寡核苷酸混合，并用高保真DNA聚合酶Pfu进行PCR反应，以产生长度类似于500 bp的DNA片段。 (ii) 合成感兴趣的 DNA 的整个序列：将第一步的 5 至 10 个 PCR 产物组合起来，并用作使用高保真 DNA 聚合酶 Pyrobest 进行第二次 PCR 反应的模板，以两个最外面的寡核苷酸作为引物。与之前发表的方法相比，PTDS方法速度快（5-7天），适合合成高G+C含量、重复序列或复杂二级结构的长DNA片段（5-6 kb）。因此，PTDS方法为合成和组装具有复杂结构的长基因提供了替代工具。利用新开发的PTDS方法，我们成功获得了多个大小在1.0至5.4 kb之间的感兴趣基因。
Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of Individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are similar to500 bp In length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5-7 days) and suitable for synthesizing long segments of DNA (5-6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.