Electrochemical detection of nucleic acid hybridization

a nucleic acid hybridization and electrochemical detection technology, applied in the field of nucleic acid hybridization and sequencing, can solve problems such as disadvantageous techniques

Inactive Publication Date: 2005-10-20
THORP H +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these techniques ate disadvantageous inasmuch as they require more time and separation technology.

Method used

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  • Electrochemical detection of nucleic acid hybridization
  • Electrochemical detection of nucleic acid hybridization
  • Electrochemical detection of nucleic acid hybridization

Examples

Experimental program
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Effect test

example 1

Measurement of Cyclic Voltammogram of Ru(bpy)32+

[0102] The cyclic voltammograms of Ru(bpy)32+ with and without calf thymus DNA are shown in FIG. 1, with the catalytic enhancement produced by the multiple turnovers of oxidation of DNA by the oxidized form of the metal complex which are observed during a single voltammetric sweep. The voltammetry of any DNA-bound redox couple must be analyzed in terms of a square-scheme that relates the bound and unbound forms because the diffusion coefficient of DNA is much lower (i.e., 2.0×10−7 cm2 / s) than that of the metal complex (8.0×10−6 cm2 / s). This phenomenon generally leads to dramatically decreased currents for the bound form; however, at sufficient high ionic strength ([Na+]=0.8 M), binding of the metal complex is too weak to influence the current response. In this case, the current can be analyzed in terms of a simple EC′ mechanism.

Ru(bpy)32+→Ru(bpy)33+  (E)

Ru(bpy)33++DNA→Ru(bpy)32++DNAox(C′)

example 2

[0103] Analysis of Cyclic Voltammograms

[0104] Cyclic voltammograms were analyzed by fitting the complete current-potential curves, with the background subtracted, using the DIGISIM™ data analysis package. The input parameters were E1 / 2 for the metal complex and the diffusion coefficients for the metal complex and the DNA, all of which were determined in separate experiments. Therefore, the sole parameter obtained from the fit was the second-order rate constant for equation 2, k=9.0×103 M−1 s−1. This same rate constant was determined over a wide range of scan rates.

[0105] The rate constant for oxidation of DNA by Ru(bpy)33+ was confirmed in two separate experiments. First, square-wave voltammograms were used to obtain a pseudo-first-order kobs for equation 2 by fitting with the COOL™ algorithm. The COOL™ algorithm uses a fitting approach that is significantly different from DIGISIM™; nevertheless, plots of kobs against DNA were linear and gave a second-order rate constant k=8.2×103...

example 3

Analysis of Cyclic Voltammograms

[0106] If the driving force for electron transfer is significantly less than the reorganizational energy (λ), a plot of RT ln k versus driving force (when corrected for work terms associated with approach of the reactants) should yield a straight line with a slope of ½. The rate constants for oxidation of DNA by a number of Metal(bpy)33+ derivatives with different redox potentials are shown in Table 1 below.

[0107] Since Marcus theory describes the driving-force dependence of the electron-transfer rate, absolute rate constants can be analyzed in terms of the following equation:

k=v exp[−β(r−r0)]exp[−(ΔG+λ)2 / 4λRT]

[0108] where v is the rate constant in the diffusion-controlled limit (1011 M−1 s−1), r is the distance between reactant and product in the activated complex, r0 is the distance of closest approach of reactant and product, and β describes the influence of the intervening medium. Incorporation of the guanine donor into the interior of the doub...

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Abstract

A method of detecting a nucleic acid (e.g., DNA, RNA) that contains at least one preselected base (e.g., adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine) comprises (a) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; (b) detecting the oxidation-reduction reaction; and (c) determining the presence or absence of the nucleic acid from the detected oxidation-reduction reaction at the preselected base. The method may be used in a variety of applications, including DNA sequencing, diagnostic assays, and quantitative analysis.

Description

[0001] This application is a continuation-in-part of copending application Ser. No. 08 / 495,817 filed Jun. 27, 1995, and is a continuation-in-part of copending application Ser. No. 60 / 016,265 filed Apr. 19, 1996, the disclosures of which are incorporated by reference herein in their entirety.FIELD OF THE INVENTION [0002] The present invention relates to nucleic acid hybridization and sequencing, and particularly to methods of qualitatively and quantitatively detecting nucleic acid hybridization and to methods of nucleic acid sequencing. BACKGROUND OF THE INVENTION [0003] The detection of individual DNA sequences in heterogenous samples of DNA provides a basis for identifying genes, DNA profiling, and novel approaches to DNA sequencing. One approach to DNA hybridization detection involves the use of surface bound DNA sequences which can be assayed using an analytical response that indicates hybridization of the surface-bound oligomer to a sequence in the heterogeneous sample. These an...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCB82Y15/00B82Y30/00C12Q1/6816C12Q1/6825C12Q1/6869C12Q2600/156C12Q2563/113C12Q2525/197C12Q2525/117
Inventor THORP, H.JOHNSTON, DEANNAPIER, MARYLOOMIS, CARSONSISTARE, MARKKIM, JINHEUNG
Owner THORP H
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