التفاصيل البيبلوغرافية
| العنوان: |
Probing Biomolecule Recognition with Electron Transfer: Electrochemical Sensors for DNA Hybridization |
| المؤلفون: |
Napier, M. E., Loomis, C. R., Sistare, M. F., Kim, J., Eckhardt, A. E., Thorp, H. H. |
| المصدر: |
Bioconjugate Chemistry; November 26, 1997, Vol. 8 Issue: 6 p906-913, 8p |
| مستخلص: |
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)32+ (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)33+. 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 μC after hybridization and of only 8 ± 5 μC 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 μC for hybridization to complementary amplicon and of only 2−10 μC after exposure to noncomplementary DNA. |
| قاعدة البيانات: |
Supplemental Index |