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Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis
Zhu, Jianming1; Wang, Jing1,3; Cheng, Kai1; Chen, Hui1; An, Ran1; Zhang, Yaping1; Komiyama, Makoto1; Liang, Xingguo1,2
2019-12-05
Source PublicationCHEMBIOCHEM
ISSN1439-4227
Pages5
Corresponding AuthorWang, Jing(wangjing2019@qdio.ac.cn) ; Liang, Xingguo(liangxg@ouc.edu.cn)
AbstractHigh-resolution melting (HRM) analysis has been improved and applied for the first time to quantitative analysis of enzymatic reactions. By using the relative ratios of peak intensities of substrates and products, the quantitativity of conventional HRM analysis has been improved to allow detailed kinetic analysis. As an example, the ligation of sticky ends through the action of T4 DNA ligase has been kinetically analyzed, with comprehensive data on substrate specificity and other properties having been obtained. For the first time, the kinetic parameters (k(obs) and apparent K-m) of sticky-end ligation were obtained for both fully matched and mismatched sticky ends. The effect of ATP concentration on sticky-end ligation was also investigated. The improved HRM method should also be applicable to versatile DNA-transforming enzymes, because the only requirement is that the products have T-m values different enough from the substrates.
Keywordenzyme catalysis high-resolution melting kinetics ligases quantitative analysis
DOI10.1002/cbic.201900489
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 ; Pharmacology & Pharmacy
WOS SubjectBiochemistry & Molecular Biology ; Chemistry, Medicinal
WOS IDWOS:000500756500001
PublisherWILEY-V C H VERLAG GMBH
Citation statistics
Cited Times:3[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/163823
Collection海洋生态与环境科学重点实验室
Corresponding AuthorWang, Jing; Liang, Xingguo
Affiliation1.Ocean Univ China, Coll Food Sci & Engn, Qingdao 266003, Peoples R China
2.Qingdao Natl Lab Marine Sci & Technol, Lab Marine Drugs & Bioprod, Qingdao 266003, Peoples R China
3.Chinese Acad Sci, Inst Oceanol, CAS Key Lab Marine Ecol & Environm Sci, Qingdao 266071, Peoples R China
Corresponding Author AffilicationKey Laboratory of Marine Ecology & Environmental Sciences, CAS
Recommended Citation
GB/T 7714
Zhu, Jianming,Wang, Jing,Cheng, Kai,et al. Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis[J]. CHEMBIOCHEM,2019:5.
APA Zhu, Jianming.,Wang, Jing.,Cheng, Kai.,Chen, Hui.,An, Ran.,...&Liang, Xingguo.(2019).Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis.CHEMBIOCHEM,5.
MLA Zhu, Jianming,et al."Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis".CHEMBIOCHEM (2019):5.
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