Assembly of Fragment Ends After PCR: An Efficient and Accurate Multi‐part DNA Assembly Method for Large DNA Sequence

Abstract

Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology. But new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. The method requires two‐round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR that inserts overlapping overhangs at both sides of each DNA fragment. The resulting products of the second PCR are then annealed and ligated with T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. We characterized its capability and limitations and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1–12 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. AFEAP cloning provides a powerful, efficient, seamless, and sequence‐independent DNA assembly tool for multiple fragments up to 12 and large DNA up to 200 kb that expands synthetic biologist's toolbox.Support or Funding InformationThis work was supported by National Modern Agriculture Industry Technique Systems (CARS‐02) to Jingao Dong and Starting Grant from Hebei Agricultural University to Fanli Zeng (grant number ZD201622).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.