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Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures
Wang, Jing1,3; Zhu, Jianming1; Wang, Chenru1; Zhou, Guangqing1; Yu, Xin1; Fan, Huijun1; An, Ran1,2; Komiyama, Makoto1; Liang, Xingguo1,2
2020-02-04
Source PublicationBIOCHEMISTRY
ISSN0006-2960
Volume59Issue:4Pages:400-406
Corresponding AuthorKomiyama, Makoto(xgsz@ouc.edu.cn) ; Liang, Xingguo(liangxg@ouc.edu.cn)
AbstractThermus thermophilus DNA ligase (Tth DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g., 65 degrees 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 degrees 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 degrees 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.
DOI10.1021/acs.biochem.9b00866
Indexed BySCI
Language英语
Funding ProjectNational Natural Science Foundation of China[31571937] ; Shandong Provincial Natural Science Foundation of China[ZR2019BC096] ; Fundamental Research Funds for the Central Universities[201812009]
WOS Research AreaBiochemistry & Molecular Biology
WOS SubjectBiochemistry & Molecular Biology
WOS IDWOS:000511509600008
PublisherAMER CHEMICAL SOC
Citation statistics
Document Type期刊论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/165353
Collection中国科学院海洋研究所
Corresponding AuthorKomiyama, Makoto; Liang, Xingguo
Affiliation1.Ocean Univ China, Qingdao, Peoples R China
2.Qingdao Natl Lab Marine Sci & Technol, Qingdao, Peoples R China
3.Chinese Acad Sci, Inst Oceanol, Qingdao, Peoples R China
First Author AffilicationInstitute of Oceanology, Chinese Academy of Sciences
Recommended Citation
GB/T 7714
Wang, Jing,Zhu, Jianming,Wang, Chenru,et al. Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures[J]. BIOCHEMISTRY,2020,59(4):400-406.
APA Wang, Jing.,Zhu, Jianming.,Wang, Chenru.,Zhou, Guangqing.,Yu, Xin.,...&Liang, Xingguo.(2020).Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures.BIOCHEMISTRY,59(4),400-406.
MLA Wang, Jing,et al."Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures".BIOCHEMISTRY 59.4(2020):400-406.
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