Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis

doi:10.1002/cbic.201900489

	Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis
	Zhu, Jianming 1; Wang, Jing 1,3; Cheng, Kai 1; Chen, Hui 1; An, Ran 1; Zhang, Yaping 1; Komiyama, Makoto 1; Liang, Xingguo 1,2
	2019-12-05
Source Publication	CHEMBIOCHEM
ISSN	1439-4227
Pages	5
Corresponding Author	Wang, Jing(wangjing2019@qdio.ac.cn) ; Liang, Xingguo(liangxg@ouc.edu.cn)
Abstract	High-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.
Keyword	enzyme catalysis high-resolution melting kinetics ligases quantitative analysis
DOI	10.1002/cbic.201900489
Indexed By	SCI
Language	英语
Funding Project	National Natural Science Foundation of China[31571937] ; Shandong Provincial Natural Science Foundation of China[ZR2019BC096] ; Fundamental Research Funds for the Central Universities[201812009]
WOS Research Area	Biochemistry & Molecular Biology ; Pharmacology & Pharmacy
WOS Subject	Biochemistry & Molecular Biology ; Chemistry, Medicinal
WOS ID	WOS:000500756500001
Publisher	WILEY-V C H VERLAG GMBH
Citation statistics	Cited Times:3[WOS] [WOS Record] [Related Records in WOS]
Document Type	期刊论文
Identifier	http://ir.qdio.ac.cn/handle/337002/163823
Collection	海洋生态与环境科学重点实验室
Corresponding Author	Wang, Jing; Liang, Xingguo
Affiliation	1.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 Affilication	Key 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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