In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites

文献类型： 外文期刊

第一作者： Tan, Yanning

作者： Tan, Yanning;Sun, Zhizhong;Sun, Xuewu;Sheng, Xiabing;Zhou, Tianshun;Liu, Ling;Mo, Yi;Jiang, Beibei;Ouyang, Ning;Yin, Xiaolin;Duan, Meijuan;Yuan, Dingyang;Yu, Dong;Tan, Yanning;Sun, Zhizhong;Sun, Xuewu;Sheng, Xiabing;Yuan, Dingyang;Yu, Dong;Sun, Zhizhong;Mo, Yi;Duan, Meijuan;Yuan, Dingyang;Zhou, Tianshun;Liu, Ling;Jiang, Beibei;Ouyang, Ning;Yin, Xiaolin;Yuan, Dingyang

作者机构：

期刊名称：SCIENTIFIC REPORTS （ 影响因子：4.379； 五年影响因子：5.133 ）

ISSN： 2045-2322

年卷期： 2017 年 7 卷

页码：

收录情况： SCI

摘要： The standard binary enzymatic assembly, which operates by inserting one DNA fragment into a plasmid, has a higher assembly success rate than the polynary enzymatic assembly, which inserts two or more fragments into the plasmid. However, it often leaves a nucleotide scar at the junction site. When a large DNA molecule is assembled stepwise into a backbone plasmid in a random piecewise manner, the scars will damage the structure of the original DNA sequence in the final assembled plasmids. Here, we propose an in vitro Seamless Stack Enzymatic Assembly (SSEA) method, a novel binary enzymatic assembly method involving a seamless strategy of splicing restriction sites via a stepwise process of multiple enzymatic reactions that does not leave nucleotide scars at the junction sites. We have demonstrated the success and versatility of this method through the assembly of 1) a 4.98 kb DNA molecule in the 5'-> 3' direction using BamHI to generate the sticky end of the assembly entrance, 2) a 7.09 kb DNA molecule in the 3'-> 5' direction using SmaI to generate the blunt end of the assembly entrance, and 3) an 11.88 kb DNA molecule by changing the assembly entrance.

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