Electrochemical detection of osmium tetroxide-labeled PCR-products by means of protective strands.
This communication reports about how single-stranded 136 base polymerase chain reaction (PCR) products labeled with electrochemically active osmium tetroxide bipyridine can be detected voltammetrically by hybridization with probe strands immobilized on gold electrodes. These electroactive ssDNA targets have been obtained by means of Lambda Exonuclease treatment of the double-stranded PCR products followed by hybridization of the remaining single strands with short protective strands and covalent labeling with osmium tetroxide bipyridine. Square-wave voltammetric signals of these osmium labels have been obtained only upon hybridization with the immobilized probe strands. An optimal 50 degrees C hybridization temperature has been found with a saturation of the probe layer at 30 min hybridization time and 7.5 nmol/l target concentration. The blank capture probe layer alone did not yield any signal. Unprotected strands produced almost no interference. Such double-selective switch-on electrochemical hybridization assays hold great promise for the specific detection of PCR products.
Reske
Thomas
T
University of Rostock, Department of Chemistry, Dr. -Lorenz-Weg 1, D-18051 Rostock, Germany.
Mix
Maren
M
Bahl
Hubert
H
Flechsig
Gerd-Uwe
GU
D015342:DNA Probes / Q000032:analysis / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000737:chemistry; D004563:Electrochemistry; D004566:Electrodes; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000737:chemistry; D009942:Organometallic Compounds / Q000032:analysis / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011725:Pyridines / Q000032:analysis / Q000737:chemistry*; D013194:Staining and Labeling; D013696:Temperature; D013997:Time Factors
D015342:DNA Probes; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D009942:Organometallic Compounds; D011725:Pyridines; C092906:osmium tetroxide bispyridine; D006046:Gold; D005090:Exodeoxyribonucleases