Thermus thermophilus DNA ligase connects two fragments having exceptionally short complementary termini at high temperatures

Thermus thermophilus DNA ligase (Tth DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g. 65°C). It has been long believed that the complementary ends must be very long (e.g. >30 bp) to place two DNA fragments nearby for the ligation. In the current study, the length of complementary portion was systematically varied, and the ligation efficiency was evaluated using high resolution melting (HRM) method. Unexpectedly, very short oligonucleotides (7-10 nt) were successfully ligated on the complementary overhang attached to a dsDNA at 70°C. Furthermore, sticky ends with the overhang of only 4 nt long, available after scission with many restriction enzymes, were also efficiently ligated at 45-70°C. The ligation yield for the 6 nt long sticky ends was as high as 80%. It was concluded that Tth DNA ligase can be used as a unique tool for DNA manipulation that cannot be otherwise easily accomplished.