Selective Dehybridization using Electrochemically-Generated Reagent on an Electrode Microarray
AssigneeCombiMatrix Corporation
InventorsJohn Cooper, Andrei Gindilis
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Abstract
The present invention provides a method for selective dehybridization by electrochemically-generated (ECG) reagent on an electrode microarray. The ECG reagent is generated by activation of selected electrodes. Activation alters pH in the vicinity of only the selected electrodes. In one embodiment, the increase or decrease in pH is sufficient to cause dehybridization of an oligonucleotide duplex at the selected electrodes. In another embodiment, the increase or decrease in pH is sufficient to prevent chemical dehybridization at the selected electrodes. The dehybridized single stranded target oligonucleotide may be recovered and amplified by PCR.
Claims28
A method for selective dehybridization by electrochemically-generated reagent on an electrode microarray comprising:
- (a) providing an electrode microarray having an lectrode surface having at least one electrode proximate to a porous reaction layer having at least one oligonucleotide duplex, wherein the at least one oligonucleotide duplex comprises a target oligonucleotide and a probe oligonucleotide, wherein a dehybridizing solution contacts the porous reaction layer and the electrode surface;
- and (b) dehybridizing the at least one oligonucleotide duplex by generating an electrochemically-generated reagent, whereby the target oligonucleotide goes into the dehybridizing solution the probe nucleotide substantially remains attached to the porous reaction layer.
The method of claim 11 wherein the buffer is selected from the group consisting of di-sodium phosphate, mono-sodium phosphate, citrate, carbonate, bicarbonate, borate, acetate, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, HEPES, TES, DIPSO, TAPSO, TRIZMA, HEPPSO, POPSO, EPPS, TEA, Tricine, Bicine, TAPS, AMPSO, CHES, CAPSO, AMP, CAPS and combinations thereof.
A method for selective dehybridization by electrochemically-generated reagent on an electrode microarray comprising:
- (a) providing an electrode microarray having an electrode surface having at least a first electrode and a second electrode, wherein the first electrode is proximate to a first porous reaction layer and the second electrode is proximate to a second porous reaction layer;
- (b) binding a first probe oligonucleotide to the first porous reaction layer and a second probe oligonucleotide to the second porous reaction layer;
- (c) hybridizing a first target oligonucleotide to the first probe oligonucleotide and a second target oligonucleotide to the second probe oligonucleotide, wherein the first target oligonucleotide and the first probe oligonucleotide form a first oligonucleotide duplex and the second target oligonucleotide and the second probe oligonucleotide form a second oligonucleotide duplex;
- and (d) dehybridizing the first oligonucleotide duplex by an electrochemically-generated reagent generated in a dehybridizing solution contacting the first porous reaction layer, the second reaction layer, and the electrode surface, wherein the electrochemically-generated reagent is generated by an activation means applied to the first electrode, whereby the first target nucleotide goes into the dehybridizing solution, the first probe oligonucleotide substantially remains attached to the first reaction layer, and the second oligonucleotide duplex is not dehybridized.
A method for selective dehybridization by electrochemically-generated reagent on an electrode microarray comprising:
- (a) providing an electrode microarray having an electrode surface having at least a first electrode and a second electrode, wherein the first electrode is proximate to a first porous reaction layer having a plurality of first oligonucleotide duplexes and the second electrode is proximate to a second porous reaction layer having a plurality of second oligonucleotide duplexes, wherein each of the plurality of first oligonucleotide duplexes comprise a first probe oligonucleotide and a first target oligonucleotide and each of the plurality of second oligonucleotide duplexes comprise a second probe oligonucleotide and a second target oligonucleotide;
- and (b) applying a dehybridization solution to the electrode microarray;
- and (c) generating an electrochemical reagent at the second electrode, whereby the electrochemically-generated reagent prevents dehybridization of the plurality of second oligonucleotide duplexes.
A method for selective dehybridization by electrochemically-generated reagent on an electrode microarray comprising:
- (a) providing an electrode microarray having an electrode surface having at least a first electrode and a second electrode, wherein the first electrode is proximate to a first porous reaction layer and the second electrode is proximate to a second porous reaction layer;
- (b) binding a first probe oligonucleotide to the first porous reaction layer and a second probe oligonucleotide to the second porous reaction layer;
- (c) hybridizing a first target oligonucleotide to the first probe oligonucleotide and a second target oligonucleotide to the second probe oligonucleotide, wherein the first target oligonucleotide and the first probe oligonucleotide form a first oligonucleotide duplex and the second target oligonucleotide and the second probe oligonucleotide form a second oligonucleotide duplex;
- (d) applying a chemical dehybridizing solution to the electrode microarray;
- and (e) generating an electrochemically-generated reagent at the second electrode and second porous reaction layer, whereby the electrochemically-generated reagent prevents dehybridization of the plurality of second oligonucleotide duplexes.
Dependencies are inferred from the claim text.