title: Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method.
abstract: Efficient assembly of multiple DNA fragments is a pivotal technology for synthetic biology. A scarless and sequence-independent DNA assembly method (DATEL) using thermal exonucleases has been developed recently. Here, we present a simplified DATEL (sDATEL) for efficient assembly of unphosphorylated DNA fragments with low cost. The sDATEL method is only dependent on Taq DNA polymerase and Taq DNA ligase. After optimizing the committed parameters of the reaction system such as pH and the concentration of Mg2+and NAD+, the assembly efficiency was increased by 32-fold. To further improve the assembly capacity, the number of thermal cycles was optimized, resulting in successful assembly 4 unphosphorylated DNA fragments with an accuracy of 75%. sDATEL could be a desirable method for routine manual and automated assembly.
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.|b Synergetic Innovation Center of Food Safety and Nutrition , Jiangnan University, Wuxi, Jiangsu, China Jiangnan University , Wuxi , China.
authors.forename: Wenwen
authors.initials: W
authors.lastname: Ding
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.|b Synergetic Innovation Center of Food Safety and Nutrition , Jiangnan University, Wuxi, Jiangsu, China Jiangnan University , Wuxi , China.
authors.forename: Huanjiao
authors.initials: H
authors.lastname: Weng
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.|b Synergetic Innovation Center of Food Safety and Nutrition , Jiangnan University, Wuxi, Jiangsu, China Jiangnan University , Wuxi , China.
authors.forename: Peng
authors.initials: P
authors.lastname: Jin
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.|b Synergetic Innovation Center of Food Safety and Nutrition , Jiangnan University, Wuxi, Jiangsu, China Jiangnan University , Wuxi , China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.|b Synergetic Innovation Center of Food Safety and Nutrition , Jiangnan University, Wuxi, Jiangsu, China Jiangnan University , Wuxi , China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.|b Synergetic Innovation Center of Food Safety and Nutrition , Jiangnan University, Wuxi, Jiangsu, China Jiangnan University , Wuxi , China.|c The Key Laboratory of Carbohydrate Chemistry and Biotechnology , Ministry of Education, Jiangnan University , Wuxi , P. R. China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Kang
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D053938:DNA Fragmentation; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D010766:Phosphorylation; D019914:Taq Polymerase / Q000737:chemistry* / Q000235:genetics*
chemical_list: D004247:DNA; D019914:Taq Polymerase; D005090:Exodeoxyribonucleases
keywords: DNA assembly; PCR; Simplified DATEL; Synthetic biology; Unphosphorylated DNA fragments