Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA.
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 E(1/2) 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 T(m) = 71 versus 75 degrees 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.
Wlassoff
Wjatschesslaw A
WA
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
King
Garry C
GC
D001483:Base Sequence; D015342:DNA Probes / Q000138:chemical synthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D004563:Electrochemistry / Q000379:methods*; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization / Q000379:methods; D010084:Oxidation-Reduction
C458220:5-(3-ferroceneacet-amidopropenyl-1) 2'-deoxyuridine 5'-triphosphate; C458219:5-(3-ferrocenecarboxamidopropenyl-1) 2'-deoxyuridine 5'-triphosphate; D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D004259:DNA-Directed DNA Polymerase