Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

被引:58
作者
Wlassoff, WA [1 ]
King, GC [1 ]
机构
[1] Univ New S Wales, Sch Biotechnol & Biomol Sci, Sydney, NSW 2052, Australia
关键词
D O I
10.1093/nar/gnf058
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2'-deoxyuridine 5'-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacetamidopropenyl-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 75degreesC 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.
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页数:7
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