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A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

文献类型： 外文期刊

作者： Xiong, AS 1 ; Yao, QH 2 ; Peng, RH 2 ; Li, X 2 ; Fan, HQ 2 ; Cheng, ZM 2 ; Li, Y 2 ;

作者机构： 1.Shanghai Acad Agr Sci, Agrobiotechnol Res Ctr, Shanghai 201106, Peoples R China

2.Shanghai Acad Agr Sci, Agrobiotechnol Res Ctr, Shanghai 201106, Peoples R China; Univ Tennessee, Dept Plant Sci, Knoxville, TN 37996 USA; Univ Connecticut, Dept Plant Sci, Storrs, CT 06269 USA

期刊名称：NUCLEIC ACIDS RESEARCH （ 影响因子：16.971； 五年影响因子：15.542 ）

ISSN： 0305-1048

年卷期： 2004 年 32 卷 12 期

页码：

收录情况： SCI

摘要： 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.

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