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Literature DB >> 18489897

A spectroscopic method to determine the activity of the restriction endonuclease EcoRV that involves a single reaction.

Abstract

A one-step protocol is presented to determine the activity of EcoRV as a model of restriction enzymes. The protocol involved a molecular beacon as DNA substrate, with the target sequence recognized by EcoRV in the stem. EcoRV cleaved the stem forming two fragments, one of which contained the fluorophore and quencher, initially bound by 3 bp. This shorter fragment rapidly dissociated at 37 °C, causing an increase of fluorescence intensity that was used to gauge the reaction kinetics. The reaction can be described using the Michaelis-Menten mechanism, and the kinetic parameters obtained were compared with literature values involving other protocols.

Entities: Chemical

Mesh：

Substances：

Year: 2008 PMID： 18489897 PMCID： PMC3191280 DOI： 10.1016/j.ab.2008.04.038

Source DB: PubMed Journal: Anal Biochem ISSN： 0003-2697 Impact factor: 3.365

15 in total

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Authors: A Jeltsch; A M Pingoud
Journal: Methods Mol Biol Date: 2001

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Authors: N Shimamoto
Journal: J Biol Chem Date: 1999-05-28 Impact factor: 5.157

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Journal: Methods Enzymol Date: 1992 Impact factor: 1.600

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Authors: A Pingoud; M Fuxreiter; V Pingoud; W Wende
Journal: Cell Mol Life Sci Date: 2005-03 Impact factor: 9.261

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Authors: S G Erskine; G S Baldwin; S E Halford
Journal: Biochemistry Date: 1997-06-17 Impact factor: 3.162

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Authors: J D Taylor; S E Halford
Journal: Biochemistry Date: 1989-07-25 Impact factor: 3.162

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Authors: A Jeltsch; A Fritz; J Alves; H Wolfes; A Pingoud
Journal: Anal Biochem Date: 1993-09 Impact factor: 3.365

8. Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease.

Authors: G S Baldwin; I B Vipond; S E Halford
Journal: Biochemistry Date: 1995-01-17 Impact factor: 3.162

9. Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases.

Authors: I B Vipond; G S Baldwin; S E Halford
Journal: Biochemistry Date: 1995-01-17 Impact factor: 3.162

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Authors: T R Waters; B A Connolly
Journal: Biochemistry Date: 1994-02-22 Impact factor: 3.162

1 in total

1. Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases.

Authors: Ines Fonfara; Ute Curth; Alfred Pingoud; Wolfgang Wende
Journal: Nucleic Acids Res Date: 2011-09-29 Impact factor: 16.971

1 in total