Ligase Fidelity and Bias in End-Joining Ligation: Enabling complex, multi-fragment Golden Gate DNA Assembly

In this webinar, you will learn about our recently developed a single-molecule sequencing method to characterize end-joining ligase fidelity (discrimination against ligating mismatched overhangs) and bias (sequence preferences) for short cohesive ends in a high throughput manner. This method allows determination of the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and sequence-dependent biases in ligation efficiency. We have applied the fidelity and bias data to optimize design of Golden Gate-type assemblies, allowing selection of overhang sets with minimized mismatch potential and high efficiency.