Probing Biomolecule Recognition with Electron Transfer: Electrochemical Sensors for DNA Hybridization
| Autor: | Jinheung Kim, Carson R. Loomis, Mark F. Sistare, Mary E. Napier, and Allen E. Eckhardt, H. Holden Thorp |
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| Rok vydání: | 1997 |
| Předmět: |
Guanine
Biomedical Engineering Pharmaceutical Science Guanosine Bioengineering Biosensing Techniques Polymerase Chain Reaction Electron Transport chemistry.chemical_compound Nucleic acid thermodynamics Electrochemistry Electrodes DNA Primers Pharmacology Oligonucleotide DNA–DNA hybridization Hybridization probe Organic Chemistry Nucleic Acid Hybridization DNA Molecular biology genomic DNA chemistry DNA Probes Biotechnology |
| Zdroj: | Bioconjugate Chemistry. 8:906-913 |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/bc9701149 |
| Popis: | Identifying infectious organisms, quantitating gene expression, and sequencing genomic DNA on chips all rely on the detection of nucleic acid hybridization. Described here is a novel assay for detection of the hybridization of products of the polymerase chain reaction using electron transfer from guanine to a transition-metal complex. The hybridization assay was modeled in solution by monitoring the cyclic voltammetry of Ru(bpy)3(2+) (bpy = 2,2'-bipyridine) in the presence of a probe strand containing only A, T, and C prior to and after hybridization to a complement that contained seven guanines, which led to high catalytic current due to the oxidation of guanine by Ru(bpy)3(3+). To allow recognition of all four bases in the target sequence, it was shown that inosine 5'-monophosphate was 3 orders of magnitude less reactive than guanosine 5'-monophosphate, suggesting that effective hybridization sensors could be realized by immobilization of probe strands in which inosine was substituted for guanosine; hybridization to guanosine-containing target strands would then provide high catalytic currents. A sensor design was tested in a model system for the detection of a synthetic 21-mer oligonucleotide patterned on the sequence of the ras oncogene, which gave an increase in charge collected of 35 +/- 5 microC after hybridization and of only 8 +/- 5 microC after exposure to noncomplementary DNA. Independent quantitation of probe and target by radiolabeling showed that the hybridized electrode contained 3.0 +/- 0.3 ng of target. New sensor electrodes were then prepared for the detection of PCR-amplified genomic DNA from herpes simplex virus type II, genomic DNA from Clostridium perfringens, and genomic RNA from human immunodeficiency virus and gave an additional charge of 35-65 microC for hybridization to complementary amplicon and of only 2-10 microC after exposure to noncomplementary DNA. |
| Databáze: | OpenAIRE |
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