In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites. Issue 1 (December 2017)

• Record Type: Journal Article
• Authors: Yu, Dong; Tan, Yanning; Sun, Zhizhong; Sun, Xuewu; Sheng, Xiabing; Zhou, Tianshun; Liu, Ling; Mo, Yi; Jiang, Beibei; Ouyang, Ning; Yin, Xiaolin; Duan, Meijuan; Yuan, Dingyang
• Is Part Of: Scientific reports. Volume 7:Issue 1(2017)
• Page Start: 1
• Page End: 10
• Publication Date: 2017-12
• Subjects: Natural history -- Research -- Periodicals; Biology -- Research -- Periodicals; Physical sciences -- Research -- Periodicals; Earth sciences -- Research -- Periodicals; Environmental sciences -- Research -- Periodicals; 502.85
• DOI: 10.1038/s41598-017-14496-5
• Languages: English
• ISSNs: 2045-2322

Abstract

Abstract 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 anin 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.