Use of Pacific Biosciences Single Molecule Sequencing to Assay DNA Ligase Fidelity
Synthetic Biology Engineering Evolution Design SEED
2015
2015 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Poster Session B
Friday, June 12, 2015 - 5:15pm to 6:45pm
DNA ligases, key enzymes in molecular biology and synthetic biology, catalyze the joining of single strand breaks in duplex DNA (nicks) as well as the joining of multiple dsDNA fragments. Ligation fidelity has important consequences for the yield, accurate assembly, and maximum number of fragments that can be assembled concurrently in synthetic biology applications such as Golden Gate cloning. In this study, we have applied Pacific Biosciences single-molecule sequencing technology to develop a new method to characterize the fidelity of ligation when joining DNA fragments with short (three or four base) 5’-overhangs. Pools of substrates containing all possible overhang sequences were ligated with T4 DNA ligase, and the ligation products were read directly by sequencing individual molecules. This work has allowed us to determine the overall mismatch ligation frequency, the mismatch frequency for each individual subsequence, and to characterize the particular mismatches that have a high propensity for ligation. This methodology is easily adaptable to other dsDNA substrate structures, and provides a method to screen for ligases or buffer conditions that provide higher fidelity ligation reaction outcomes. The information generated by this study will allow the optimization of DNA fragment assembly protocols, both by the development of high fidelity ligases and by the avoidance of non-complementary overhang pairs prone to misligation.