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Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures.
Biochemistry ( IF 2.9 ) Pub Date : 2020-01-10 , DOI: 10.1021/acs.biochem.9b00866
Jing Wang 1, 2 , Jianming Zhu 1 , Chenru Wang 1 , Guangqing Zhou 1 , Xin Yu 1 , Huijun Fan 1 , Ran An 1, 3 , Makoto Komiyama 1 , Xingguo Liang 1, 3
Affiliation  

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 the complementary portion was systematically varied, and the ligation efficiency was evaluated using the 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.

中文翻译:

嗜热栖热菌DNA连接酶在高温下连接两个具有极短互补末端的片段。

嗜热栖热菌DNA连接酶(Tth DNA连接酶)被广泛用于高温(例如65°C)的克隆,酶促合成和分子诊断。长期以来,人们一直认为互补末端必须非常长(例如,> 30 bp),才能将两个DNA片段放置在附近以进行连接。在当前的研究中,互补部分的长度被系统地改变,并且使用高分辨率熔解(HRM)方法评估了连接效率。出乎意料的是,在70°C下,非常短的寡核苷酸(7-10 nt)成功连接到与dsDNA相连的互补突出端。此外,在用许多限制性酶切开后可获得的仅具有4nt长突出端的粘性末端在45-70℃下也有效连接。6nt长的粘性末端的连接产率高达80%。
更新日期:2020-01-10
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