Multiply osmium-labeled reporter probes for electrochemical DNA hybridization assays: detection of trinucleotide repeats.
In electrochemical DNA hybridization assays target or probe DNAs end-labeled with electroactive compounds have been frequently used. We show that multiple osmium labels yielding faradaic (at carbon or mercury electrodes) and catalytic signals (at mercury electrodes) can be easily covalently bound to DNA molecules. We use (GAA)(7) (T)(n) oligodeoxynucleotides (ODNs) with n ranging between 5 and 50. (T)(n) tails are selectively modified with osmium tetroxide,2,2'-bipyridine leaving the (GAA)(7) repeat intact for the DNA hybridization. These ODNs are applied as reporter probes (RP's) in DNA hybridization double-surface (DS) assay using magnetic beads for the DNA hybridization and pyrolytic graphite (PGE) or hanging mercury drop (HMDE) electrodes for the electrochemical detection. We show that in difference to the usual single-surface methods (where the RP has to be bound to target DNA near to the surface to communicate with the electrode) in the DS assay the RP can be bound to DNA regardless of its position and can used for the determination of the length of DNA repetitive sequences. Several fmols or about a hundred of amol of a RP with osmium-labeled (T)(50) tail can be detected at PGE and HMDE, respectively, at 1-2 min accumulation time.
Fojta
Miroslav
M
Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, CZ-612 65 Brno, Czech Republic. fojta@ibp.cz
Havran
Ludek
L
Kizek
Rene
R
Billova
Sabina
S
Palecek
Emil
E
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D017930:Genes, Reporter; D017403:In Situ Hybridization / Q000295:instrumentation / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000032:analysis* / Q000737:chemistry*; D009992:Osmium / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods; D018911:Trinucleotide Repeats
D020099:Coated Materials, Biocompatible; D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; D009992:Osmium; D004247:DNA