Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer.

Abstract:

:The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of fluorophor-labeled oligonucleotide substrates have been examined using fluorescence resonance energy transfer (FRET). A series of duplex substrates were synthesized with an internal CTCGAG PaeR7 recognition site and donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescence resulting from restriction cleavage of these substrates was continuously monitored and the initial rate data was fitted to the Michaelis-Menten equation. The steady state kinetic parameters for these substrates were in agreement with the rate constants obtained from a gel electrophoresis-based fixed time point assay using radiolabeled substrates. The FRET method provides a rapid continuous assay as well as high sensitivity and reproducibility. These features should make the technique useful for the study of DNA-cleaving enzymes.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Ghosh SS,Eis PS,Blumeyer K,Fearon K,Millar DP

doi

10.1093/nar/22.15.3155

subject

Has Abstract

pub_date

1994-08-11 00:00:00

pages

3155-9

issue

15

eissn

0305-1048

issn

1362-4962

journal_volume

22

pub_type

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