Rapid and accurate assembly of large DNA assisted by in vitro packaging of bacteriophage

Abstract

AbstractDevelopment of DNA assembly methods made it possible to construct large DNA. However, achieving the large DNA assembly easily, accurately, and at 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 multiple PCR fragments using the bacteriophage in vitro packaging system. This method, named 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.Abstract Figure