Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage

[Image: see text] Development of DNA assembly methods made it possible to construct large DNA. However, achieving a large DNA assembly easily, accurately, and at a low cost remains a challenge. This study shows that DNA assembled only by annealing of overlapping single-stranded DNA ends, which are generated by exonuclease treatment, without ligation can be packaged in phage particles and can also be transduced into bacterial cells. Based on this, I developed a simple method to construct long DNA of about 40–50 kb from five to ten PCR fragments using the bacteriophage in vitro packaging system. This method, namely, iPac (in vitroPackaging-assisted DNA assembly), allowed accurate and rapid construction of large plasmids and phage genomes. This simple method will accelerate research in molecular and synthetic biology, including the construction of gene circuits or the engineering of metabolic pathways.