DICP OpenIR
Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction
Li, Xiaomin; Song, Chen; Zhao, Meiping; Li, Yuanzong
KeywordUniversal Molecular Beacon Real-time Polymerase Chain Reaction Restriction Endonucleases
Source PublicationANALYTICAL BIOCHEMISTRY
2008-10-01
DOI10.1016/j.ab.2008.06.027
Volume381Issue:1Pages:1-7
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Physical Sciences
WOS SubjectBiochemical Research Methods ; Biochemistry & Molecular Biology ; Chemistry, Analytical
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS KeywordECO-RI ENDONUCLEASE ; DNA CLEAVAGE ; RECOGNITION SITE ; SYNTHETIC OLIGODEOXYNUCLEOTIDES ; FLUOROMETRIC ASSAY ; SEQUENCES FLANKING ; FLUORESCENCE ; KINETICS ; MICROCHIP
AbstractWe describe a method for sensitive monitoring of restriction endonuclease kinetics and activity by use of a universal molecular beacon (U-MB) coupled with real-time polymerase chain reaction (PCR). The method is used to monitor the progress of DNA cleavage in a sealed reaction tube and offers more accurate and high-throughput detection. The template has a universal tail hybridized with the U-MB and the remaining sequence is complementary to one of the restriction endonuclease digestion products. The U-MB is replaced by the extension of digested product and the fluorescence quenches. With this concept, one universal fluorescence probe can be used in different enzyme analytical systems. In the work described here, homogenous assays were performed with the restriction endonucleases AluI, EcoRI, XhoI, and Sacl at smoothly controlled temperature. Cleavage efficiencies were determined, and the potential applications of this method were discussed. Furthermore, the AluI and EcoRI cleavage reactions were monitored online at varying substrate concentrations at the molecular level, and K(m), V(max), and K(cat) Values were calculated. The results suggest that U-MB monitoring of restriction endonuclease assays based on real-time PCR will be very useful for high-throughput, sensitive, and precise assays for enzyme activity screening and evolutionary biotechnology analysis. (c) 2008 Elsevier Inc. All rights reserved.
Language英语
WOS IDWOS:000258729700001
Citation statistics
Cited Times:12[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/141064
Collection中国科学院大连化学物理研究所
AffiliationPeking Univ, BNLMS, Key Lab Bioorgan Chem & Mol Engn, Coll Chem & Mol Engn, Beijing 100871, Peoples R China
Recommended Citation
GB/T 7714
Li, Xiaomin,Song, Chen,Zhao, Meiping,et al. Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction[J]. ANALYTICAL BIOCHEMISTRY,2008,381(1):1-7.
APA Li, Xiaomin,Song, Chen,Zhao, Meiping,&Li, Yuanzong.(2008).Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction.ANALYTICAL BIOCHEMISTRY,381(1),1-7.
MLA Li, Xiaomin,et al."Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction".ANALYTICAL BIOCHEMISTRY 381.1(2008):1-7.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Google Scholar
Similar articles in Google Scholar
[Li, Xiaomin]'s Articles
[Song, Chen]'s Articles
[Zhao, Meiping]'s Articles
Baidu academic
Similar articles in Baidu academic
[Li, Xiaomin]'s Articles
[Song, Chen]'s Articles
[Zhao, Meiping]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[Li, Xiaomin]'s Articles
[Song, Chen]'s Articles
[Zhao, Meiping]'s Articles
Terms of Use
No data!
Social Bookmark/Share
All comments (0)
No comment.
 

Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.