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.

Keywords: DNA assembly; PCR; Simplified DATEL; Synthetic biology; Unphosphorylated DNA fragments.

Figure 1.

Principle and optimization of sDATEL assembly method. (A) Schematic diagram of sDATEL. The length of overhang was set as 30 nt while the primers for amplifying DNA fragments were designed with 40 nt. After denaturation and annealing, the displaced overhangs at the fork structures were cleaved by Taq DNA polymerase and the nicks were joined by Taq DNA ligase; (B) Schematic diagram of DATEL; (C), (D) and (E) were the effects on the assembly efficiency that caused by pH, NAD+ and Mg²⁺, respectively.

Figure 2.

Performance of sDATEL for assembling multiple fragments. (A) Schematic diagram of the assembly of 2, 3 and 4 DNA fragments; (B), (C) and (D) were the gel electrophoresis analysis results for assembly of 2, 3 and 4 DNA fragments, respectively, and (E), (F) and (G) were the corresponding plates.

Figure 3.

Assembly capacity and accuracy of sDATEL method.