Abstract
DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method’s utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.
Highlights
Advances in techniques to manipulate DNA ignited biology’s genetic revolution and have underpinned modern advances in biomedical science [1]
Binding of the Scaffold Oligonucleotide Connectors (SOCs) to the complementary strands of the two fragments connects them for assembly, creating a temporary dsDNA structure with a single phosphodiester bond missing between the two complementary strands (Figure 1C)
The ideal DNA assembly technique for a given cloning project depends on a number of factors such as the complexity of the desired construct, sizes of fragments to be assembled, rates of throughput needed, experience with molecular biology, and cost constraints
Summary
Advances in techniques to manipulate DNA ignited biology’s genetic revolution and have underpinned modern advances in biomedical science [1]. Techniques to assemble the amplified DNA sequences into circular plasmids for downstream applications have for many years been mostly limited to restriction digest-based assemblies [3]. Restriction endonucleases are DNAcleaving enzymes that bind and create double-stranded (ds) DNA breaks at specific palindromic sequences, enabling cut DNA fragments to be joined using a DNA ligase into vectors digested with the same endonuclease [3]. Ligation, transformation, isolation, and sequencing are required for even the most routine multi-insert constructs. Ligated restriction fragments often have ‘‘scar’’ sequences, unwanted nucleotides left behind at joining sites, limiting user control over the final sequence. This is especially problematical, for example, when a protein-coding sequence must be preserved
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