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

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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660187/
https://www.ncbi.nlm.nih.gov/pubmed/29079784
http://dx.doi.org/10.1038/s41598-017-14496-5
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author 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
author_facet 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
author_sort Yu, Dong
collection PubMed
description 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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spelling pubmed-56601872017-11-01 In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites 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 Sci Rep Article 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. Nature Publishing Group UK 2017-10-27 /pmc/articles/PMC5660187/ /pubmed/29079784 http://dx.doi.org/10.1038/s41598-017-14496-5 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
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
In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites
title In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites
title_full In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites
title_fullStr In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites
title_full_unstemmed In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites
title_short In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites
title_sort in vitro seamless stack enzymatic assembly of dna molecules based on a strategy involving splicing of restriction sites
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660187/
https://www.ncbi.nlm.nih.gov/pubmed/29079784
http://dx.doi.org/10.1038/s41598-017-14496-5
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