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Efficient assembly of very short oligonucleotides using T4 DNA Ligase

Daniel R Horspool1, Robin JN Coope1 and Robert A Holt12*

Author Affiliations

1 British Columbia Cancer Agency, Genome Sciences Centre, Suite 100, 570 West 7th Avenue, Vancouver, British Columbia V5Z 4S6, Canada

2 Simon Fraser University, Department of Molecular Biology and Biochemistry, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada

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BMC Research Notes 2010, 3:291  doi:10.1186/1756-0500-3-291

Published: 9 November 2010

Abstract

Background

In principle, a pre-constructed library of all possible short oligonucleotides could be used to construct many distinct gene sequences. In order to assess the feasibility of such an approach, we characterized T4 DNA Ligase activity on short oligonucleotide substrates and defined conditions suitable for assembly of a plurality of oligonucleotides.

Findings

Ligation by T4 DNA Ligase was found to be dependent on the formation of a double stranded DNA duplex of at least five base pairs surrounding the site of ligation. However, ligations could be performed effectively with overhangs smaller than five base pairs and oligonucleotides as small as octamers, in the presence of a second, complementary oligonucleotide. We demonstrate the feasibility of simultaneous oligonucleotide phosphorylation and ligation and, as a proof of principle for DNA synthesis through the assembly of short oligonucleotides, we performed a hierarchical ligation procedure whereby octamers were combined to construct a target 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.