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题名: Singly labeled smart probes for real-time monitoring of the kinetics of dNTP misincorporation and single nucleotide extension in DNA intra-molecular polymerization
作者: Song, Chen1;  Zhang, Chen1;  Zhao, Meiping1
关键词: Singly labeled smart probe ;  Polymerase ;  Klenow fragment ;  Misincorporation ;  Single nucleotide extension
刊名: BIOSENSORS & BIOELECTRONICS
发表日期: 2009-10-15
DOI: 10.1016/j.bios.2009.07.002
卷: 25, 期:2, 页:301-305
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine ;  Physical Sciences
类目[WOS]: Biophysics ;  Biotechnology & Applied Microbiology ;  Chemistry, Analytical ;  Electrochemistry ;  Nanoscience & Nanotechnology
研究领域[WOS]: Biophysics ;  Biotechnology & Applied Microbiology ;  Chemistry ;  Electrochemistry ;  Science & Technology - Other Topics
英文摘要: In this paper, a simple and rapid method was developed for real-time monitoring of the kinetics of dNTP misincorporation and single nucleotide extension in DNA intra-molecular polymerization by using singly labeled fluorophore-oligonucleotide smart probes. The probes are designed with a self-complementary T-end and a sequence of stacked cytosines at the 5'-end, to which a fluorescein (FAM) is attached. When the DNA polymerase is introduced, it will bind to the 3'-end of the probe and catalyze the extension reaction, resulting in the formation of stacked guanines, which will instantly quench the fluorescence of FAM through photoelectron transfer. The method can accurately quantify the activity of the Klenow fragment of Escherichia coli DNA polymerase I with the exonuclease activity inactivated (KF(-)) in 3 min with a detection limit down to 3.7 pM, which is much faster and more sensitive than the existing technology in monitoring the polymerization in bulk reaction. Moreover, the smart probes could be used to determine the kinetics of dNTP misincorporation and single nucleotide extension by proper design of the sequence. The method is universally adaptive to any fluorescence spectrometer and offers a very convenient and cost-effective way for characterization of the fine kinetic procedures in DNA polymerization. (C) 2009 Elsevier B.V. All rights reserved.
关键词[WOS]: ESCHERICHIA-COLI ;  KLENOW FRAGMENT ;  POLYMERASE-I ;  REVERSE-TRANSCRIPTASE ;  ELECTRON-TRANSFER ;  MOLECULAR BEACON ;  FLUORESCENT DYES ;  ASSAY ;  RESIDUES
语种: 英语
WOS记录号: WOS:000270764300006
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/141276
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Peking Univ, BNLMS, Key Lab Bioorgan Chem & Mol Engn, Minist Educ,Coll Chem & Mol Engn, Beijing 100871, Peoples R China

Recommended Citation:
Song, Chen,Zhang, Chen,Zhao, Meiping. Singly labeled smart probes for real-time monitoring of the kinetics of dNTP misincorporation and single nucleotide extension in DNA intra-molecular polymerization[J]. BIOSENSORS & BIOELECTRONICS,2009,25(2):301-305.
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