Efficient Assembly of Very Short Oligonucleotides Using T4 DNA Ligase

Background: In principle, a pre-constructed library of all possible short oligonucleotides could be used toconstruct many distinct gene sequences. In order to assess the feasibility of such an approach, we characterized T4DNA Ligase activity on short oligonucleotide substrates and defined conditions suitable for assembly of a pluralityof oligonucleotides.Findings: Ligation by T4 DNA Ligase was found to be dependent on the formation of a double stranded DNAduplex of at least five base pairs surrounding the site of ligation. However, ligations could be performed effectivelywith overhangs smaller than five base pairs and oligonucleotides as small as octamers, in the presence of asecond, complementary oligonucleotide. We demonstrate the feasibility of simultaneous oligonucleotidephosphorylation and ligation and, as a proof of principle for DNA synthesis through the assembly of shortoligonucleotides, we performed a hierarchical ligation procedure whereby octamers were combined to construct atarget 128-bp segment of the beta-actin gene.Conclusions: Oligonucleotides as short as 8 nucleotides can be efficiently assembled using T4 DNA Ligase. Thus,the construction of synthetic genes, without the need for custom oligonucleotide synthesis, appears feasible.