Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

WLASSOFF, W. A. and KING, G. C. (2002). Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA. Nucleic acids research, 30 (12).

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Official URL: http://www.ingentaconnect.com/content/oup/nar/2002...

Abstract

Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2'-deoxyuridine 5'-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacet-amidopropenyl-1) 2'-deoxyuridine 5'-triphosphate (Fc2-dUTP) have been produced to demonstrate the incorporation of redox labels into DNA by polymerases. Cyclic voltammetry indicates that the ferrocenyl moieties display reversible redox behaviour in aqueous buffer with E1/2 values of 398 (Fc1-dUTP) and 260 mV (Fc2-dUTP) versus Ag/AgCl. Primer extension by the proofreading enzymes Klenow fragment and T4 DNA polymerase shows that Fc1-dUTP is efficiently incorporated into DNA during synthesis, including incorporation of two successive modified nucleotides. Production of a 998 bp amplicon by Tth DNA polymerase demonstrates that Fc1-dUTP is also a satisfactory substrate for PCR. Despite its structural similarity, Fc2-dUTP acts predominantly as a terminator with the polymerases employed here. UV melting analysis of a 37mer duplex containing five Fc1-dU residues reveals that the labelled nucleotide introduces only a modest helix destabilisation, with Tm = 71 versus 75°C for the corresponding natural construct. Modified DNA is detected at femtomole levels using a HPLC system with a coulometric detector. The availability of simple and effective enzymatic labelling strategies should promote the further development of electrochemical detection in nucleic acid analysis.

Item Type: Article
Research Institute, Centre or Group: Biomedical Research Centre
Depositing User: Ann Betterton
Date Deposited: 04 Mar 2008
Last Modified: 09 Dec 2009 18:23
URI: http://shura.shu.ac.uk/id/eprint/404

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