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PCR-based accurate synthesis of long DNA sequences

文献类型: 外文期刊

作者: Xiong, Ai-Sheng 1 ; Yao, Quan-Hong 2 ; Peng, Ri-He 2 ; Duan, Hui 2 ; Li, Xian 3 ; Fan, Hui-Qin; Cheng, Zong-Ming; L 1 ;

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

2.Shanghai Acad Agr Sci, Agro Biotechnol Res Ctr, Shanghai 201106, Peoples R China; Univ Connecticut, Dept Plant Sci, Storrs, CT 06269 USA; Univ Illinois, Dept Cell & Struct Biol, Urbana, IL 61801 USA; Univ Tennessee, Dept Plant Sci, Knoxville, TN 37996 USA

3.Shanghai Acad Agr Sci, Agro Biotechnol Res Ctr, Shanghai 201106, Peoples R China; Univ Connecticut, Dept Plant Sci, Storrs, CT 06269 USA; Univ Illinois, Dept Cell & Struct

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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