Chemical ligation of oligodeoxyribonucleotides on circular DNA templates
by Selvasekaran, J.; Turnbull, K. D.
We report the use of small circular DNA as a triplex-directing template for the highly efficient chemical ligation of oligodeoxyribonucleotides (ODNs) using cyanogen bromide (BrCN), These investigations compared the use of a linear homopyrimidine DNA template (17mer) and a circular pyrimidine-rich DNA template (44mer) for directing the chemical ligation of two homopurine ODNs (6mer + 11mer). The effects of substrate/template ratio, buffer, salt, ionic strength, pH and temperature have been examined in the BrCN activated ligation reactions, The optimal yield of 51% for ligation on the linear template was at PR 6.0, 200 mM MgCl2, 4 degrees C, In contrast, near quantitative ligation on the circular template occurred at higher pH, higher temperature, and showed less dependence on Mg2+ concentration (97% yield, pH 7.5, 200 mM MgCl2, 25 degrees C), The relative observed rate of the ligation reaction was a minimum of 35 times faster on the circular DNA template relative to the linear template at pH 7.5, 200 mM MgCl2, 4 degrees C, These investigations reveal that chemical ligation of short ODNs on circularized DNA templates through tripler formation is a highly efficient process over a broad range of conditions.
- Journal
- Nucleic Acids Research
- Volume
- 27
- Issue
- 2
- Year
- 1999
- Start Page
- 624-627
- URL
- https://dx.doi.org/10.1093/nar/27.2.624
- ISBN/ISSN
- 1362-4962; 0305-1048
- DOI
- 10.1093/nar/27.2.624