High-efficiency DNA ligation is vital for many molecular biology experiments, and it is best achieved using reactants with non-palindromic sticky ends to maximize specificity. However, optimizing such multi-parametric ligation reactions often involves extensive trial and error. We have developed a freely available Web-based ligation calculator, NP-Sticky (http://sarkarlab. umn.edu/npsticky/), that predicts product distribution for given reactant concentrations, thus enabling straightforward computational optimization of these reactions. Built-in schemes include two-piece and three-piece linear ligation, as well as insert-vector circular ligation. The only parameters needed for the underlying thermodynamic model are the free energies of ligation for each sticky end, which can be estimated by the calculator from the overhang sequences or provided by the user from direct experimental measurement. Free energies of sticky-end mismatches are also calculated for determining the extent of byproduct formation. This ligation calculator allows rapid identification of the optimal conditions for maximizing incorporation, efficiency, and/or accuracy, based on specific needs.
Bibliographical noteFunding Information:
The authors thank Najaf Shah and Dan McDonald for helpful discussions. This work was supported by a CAREER Award from the National Science Foundation ( #CBET-1055231 ) to C.A.S.
Copyright 2020 Elsevier B.V., All rights reserved.
- DNA ligation
- Web server
- free energy of ligation
- mathematical model
- non-palindromic sticky ends