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Digestion-ligation based and recombination based methods for constructing recombinant DNA are the basic techniques in molecular biology and thus built one of the foundations for the modern life sciences. Here we describe a new strategy that can radically simplify some of the same task. The lego DNA assembling, based on strand annealing, allows in vitro assembly of multi DNA fragments in one step with precise junctions and excludes the need for any enzyme. As a proof of concept, we rapidly constructed plasmids from 4, 6 and 8 fragments with very high efficiencies (100%). And we found this method a powerful tool for synthetic biology, constructing a partial isoprene biosynthesis pathway (consisting of four genes) in 2 days. We also assembled a customized expression vector to show its modularity.

Lego DNA assembling, a simple in vitro method for constructing DNA molecule