PCR-based accurate synthesis of long DNA sequences

文献类型： 外文期刊

第一作者： Xiong, Ai-Sheng

作者： Xiong, Ai-Sheng;Yao, Quan-Hong;Peng, Ri-He;Duan, Hui;Li, Xian;Fan, Hui-Qin;Cheng, Zong-Ming;Li, Yi

作者机构：

期刊名称：NATURE PROTOCOLS （ 影响因子：13.491； 五年影响因子：17.24 ）

ISSN： 1754-2189

年卷期： 2006 年 1 卷 2 期

页码：

收录情况： SCI

摘要： Here we describe a simple and rapid method for assembly and PCR-based accurate synthesis (PAS) of long DNA sequences. The PAS protocol involves the following five steps: (i) design of the DNA sequence to be synthesized and of 60-bp overlapping oligonucleotides to cover the entire DNA sequence; (ii) purification of the oligonucleotides by PAGE; (iii) first PCR, to synthesize DNA fragments of 400-500 bp in length using 10 inner (template) and two outer (primer) oligonucleotides; (iv) second PCR, to assemble the products of the first PCR into the full-length DNA sequence; and (v) cloning and verification of the synthetic DNA by sequencing and, if needed, error correction using an overlap-extension PCR technique. This method, which takes similar to 1 wk, is suitable for synthesizing diverse types of long DNA molecule. We have successfully synthesized DNA fragments from 0.5 to 12.0 kb, with high G+C content, repetitive sequences or complex secondary structures. The PAS protocol therefore provides a simple, rapid, reliable and relatively inexpensive method for synthesizing long, accurate DNA sequences.

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