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Nucleic Acids Research, 1985, Vol. 13, No. 9 3261-3271
© 1985

Articles

Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol

Ken'ichiro Hayashi, Masako Nakazawa, Yukuo Ishizaki and Akira Obayashi

Takara Shuzo Co., Ltd., Central Research Laboratories 3-4-1 Seta, Otsu-shi, Shiga 520-21, Japan

Received February 4, 1985. Revised March 29, 1985. Accepted March 29, 1985.

Monovalent cations such as Na+ and K+ inhibit the activity of T4 DNA ligase. However, the extent of inhibition varies with the terminal sequence of the duplex DNA used as substrate; in many cases, ligation of DNA is completely inhibited at 200 mM. The activity of the ligase is stimulated by raising the concentration of polyethylene glycol 6000 from 0 to 15% (w/v) when NaCl and KCl were both absent. Ligation was reduced as the concentration of NaCl or KCl was raised in a mixture containing 5 or 15% PEG 6000. With 10% PEG 6000, both cohesive- and blunt-end ligation of this ligase increased at high concentrations of salt (150–200 mM NaCl, or 200–250 mM KCl). Further, with 10% PEG 6000, inter- and intramolecular ligation occurred at low salt concentrations (0–100 mM NaCl, or 0–150 mM KCl); only linear oligomers were formed by intermolecular ligation at the high concentrations.


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