Electrochemical detection of single base extension

Abstract

This invention relates to apparatus and methods for detecting single base extension to an oligonucleotide array using electrochemical labels.

What we claim is:  
     
         1 . An apparatus for detecting single base extension of an oligonucleotide comprising an oligonucleotide array, wherein extension is effected by a polymerase and directed by a nucleotide sequence of a nucleic acid in a biological sample, the apparatus comprising 
 a first electrode comprising an array of oligonucleotides on a substrate, wherein the electrode comprises a conducting or semiconducting surface,    a second, counter electrode comprising a conducting metal in contact with an aqueous electrolyte solution, and    a third reference electrode in contact with the aqueous electrolyte solution,    wherein each of the electrodes is electrically connected to a voltage source, and wherein the apparatus further comprises    a reaction chamber containing a polymerase and a hybridization solution comprising an electrolyte, wherein each of the electrodes is in electrochemical contact therewith, the solution further containing 
 a plurality of primer extension units comprising chain-terminating nucleotide species, wherein each different chain-terminating nucleotide species is labeled with a distinguishable electrochemical label capable of participating in a reduction/oxidation reaction at the surface of the first electrode under conditions whereby an electrical potential is applied to the electrodes, wherein each of the labeled chain-terminating nucleotide species has a specific reduction/oxidation potential, wherein a current is produced in the apparatus when a biological sample comprising a nucleic acid that hybridizes to an oligonucleotide contained in the oligonucleotide array is incubated in the reaction chamber under moderate to high stringency hybridization conditions and the nucleotide sequence of said hybridized oligonucleotide is extended by the incorporation of at least one of the chain-terminating nucleotide and a voltage is applied to the electrodes at a potential specific for the reduction/oxidation potential of the electrochemical label.  
   
     
     
         2 . The apparatus of  claim 1  wherein the oligonucleotide array is an addressable array, and wherein the first electrode comprises a plurality of electrodes corresponding to each address of said addressable array, wherein a current is produced at a particular address of said addressable array after single base extension of an oligonucleotide at said address of the array with a chain terminating nucleotide species labeled with an electrochemical reporter when a voltage is applied to the electrodes at a potential specific for the reduction/oxidation potential of the electrochemical label.  
     
     
         3 . The apparatus of  claim 1  wherein the chain-terminating nucleotide species are labeled with a transition metal complex.  
     
     
         4 . An apparatus according to  claim 3 , wherein the transition metal complex contains a transition metal ion that is ruthenium, cobalt, iron or osmium.  
     
     
         5 . An apparatus according to  claim 4 , wherein the reference electrode is an aqueous saturated calomel reference electrode and the transition metal complex is selected from the group consisting of 1,4-benzoquinone, ferrocene, tetracyanoquinodimethane, N,N,N′,N′-tetramethyl-p-phenylenediamine and tetrathiafulvalene.  
     
     
         6 . An apparatus according to  claim 4 , wherein the reference electrode is a silver/silver chloride reference electrode and the transition metal complex is selected from the group consisting of 9-aminoacridine, acridine orange, aclarubicin, daunomycin, doxorubicin, pirarubicin, ethidium bromide, ethidium monoazide, chlortetracycline, tetracycline, minocycline, Hoechst 33258, Hoechst 33342, 7-aminoactinomycin D, Chromomycin A 3 , mithramycin A, vinblastine, rifampicin, Os(bipyridine) 2 (dipyridophenazine) 2+ , Co(bipyridine) 3   3+  and Fe-bleomycin.  
     
     
         7 . An apparatus according to  claim 1 , wherein the chain-terminating nucleotide species are dideoxyribonucleotide species.  
     
     
         8 . An apparatus according to  claim 7  wherein the chain terminating nucleotide species is dideoxyadenosine, dideoxyguanine, dideoxyinosine, dideoxyxanthine, dideoxycytosine, dideocythymidine or dideoxyuracil.  
     
     
         9 . An apparatus according to  claim 1 , wherein the chain-terminating nucleotide species are acyclonucleotide species.  
     
     
         10 . An apparatus according to  claim 9 , wherein the chain-terminating species is acycloguanosine, acycloadenosine, acycloinosine, acycloxanthine, acyclocytosine, acyclothymidine or acyclouracil.  
     
     
         11 . An apparatus according to  claim 1 , wherein the chain-terminating nucleotide species are covalently linked to the electrochemical labels using a linker moiety.  
     
     
         12 . An apparatus according to  claim 11 , wherein the linker species has length of from about 10 to about 20 Angstroms.  
     
     
         13 . An apparatus according to  claim 11  selected from the group consisting of a hydrocarbon chain (CH 2 ) n , wherein n is 1 to 20, and a hydrocarbon chain further comprising an ether moiety, an ester moiety, a carboxyamide, or thioether moiety or a combination thereof.  
     
     
         14 . An apparatus according to  claim 11  wherein the linker is a siloxane.  
     
     
         15 . An apparatus according to  claim 1  wherein the polymerase is a thermostable polymerase.  
     
     
         16 . A method for detecting single base extension of an oligonucleotide comprising an oligonucleotide array, wherein extension is effected by a polymerase and directed by a nucleotide sequence of a nucleic acid in a biological sample, using an apparatus according to  claim 1 , the method comprising the steps of: 
 a) adding a biological sample containing a nucleic acid that hybridizes to an oligonucleotide contained in the oligonucleotide array comprising the first electrode to the reaction chamber that contains a hybridization buffer, a polymerase and a plurality of chain-terminating nucleotide species labeled with electrochemical reporters, wherein the reaction chamber is in electrochemical contact with each of the electrodes of the apparatus and each of the electrodes is electrically connected to a voltage source;    b) incubating the reaction chamber at a time and a temperature sufficient to permit single base extension of an oligonucleotide comprising the oligonucleotide array with at least one of the labeled chain-terminating nucleotide species; and    c) producing a current in the first electrode by applying a potential equal to the reduction/oxidation potential of the labeled chain-terminating nucleotide species incorporated into the oligonucleotide.    
     
     
         17 . A method according to  claim 16 , wherein the oligonucleotide array is an addressable array, and wherein the first electrode comprises a plurality of electrodes corresponding to each address of said addressable array, wherein a current is produced at a particular address of said addressable array after single base extension of an oligonucleotide at said address of the array by applying a potential equal to the reduction/oxidation potential of the chain-terminating nucleotide species labeled with electrochemical reporters incorporated into the oligonucleotide.  
     
     
         18 . The method of  claim 16  wherein the chain-terminating nucleotide species are labeled with a transition metal complex.  
     
     
         19 . An apparatus according to  claim 18 , wherein the transition metal complex contains a transition metal ion that is ruthenium, cobalt, iron or osmium.  
     
     
         20 . An apparatus according to  claim 19 , wherein the reference electrode is an aqueous saturated calomel reference electrode and the transition metal complex is selected from the group consisting of 1,4-benzoquinone, ferrocene, tetracyanoquinodimethane, N,N,N′,N′-tetramethyl-p-phenylenediamine and tetrathiafulvalene.  
     
     
         21 . An apparatus according to  claim 19 , wherein the reference electrode is a silver/silver chloride reference electrode and the transition metal complex is selected from the group consisting of 9-amino acridine, acridine orange, aclarubicin, daunomycin, doxorubicin, pirarubicin, ethidium bromide, ethidium monoazide, chlortetracycline, tetracycline, minocycline, Hoechst 33258, Hoechst 33342, 7-aminoactinomycin D, Chromomycin A 3 , mithramycin A, vinblastine, rifampicin, Os(bipyridine) 2 (dipyridophenazine) 2+ , Co(bipyridine) 3   3+  and Fe-bleomycin.  
     
     
         22 . An apparatus according to  claim 16 , wherein the chain-terminating nucleotide species are dideoxyribonucleotide species.  
     
     
         23 . An apparatus according to  claim 22  wherein the chain terminating nucleotide species is dideoxyadenosine, dideoxyguanine, dideoxyinosine, dideoxyxanthine, dideoxycytosine, dideocythymidine or dideoxyuracil.  
     
     
         24 . An apparatus according to  claim 16 , wherein the chain-terminating nucleotide species are acyclonucleotide species.  
     
     
         25 . An apparatus according to  claim 24 , wherein the chain-terminating species is acycloguanosine, acycloadenosine, acycloinosone, acyclocytosine, acyclothymidine or acyclouracil.  
     
     
         26 . An apparatus according to  claim 16 , wherein the chain-terminating nucleotide species are covalently linked to the electrochemical labels using a linker moiety.  
     
     
         27 . An apparatus according to  claim 26 , wherein the linker species has length of from about 10 to about 20 Angstroms.  
     
     
         28 . An apparatus according to  claim 26  selected from the group consisting of a hydrocarbon chain (CH 2 ) n , wherein n is 1 to 20, and a hydrocarbon chain further comprising an ether moiety, an ester moiety, a carboxyamide, or thioether moiety or a combination thereof.  
     
     
         29 . An apparatus according to  claim 26  wherein the linker is a siloxane.  
     
     
         30 . An apparatus according to  claim 16  wherein the polymerase is a thermostable polymerase.  
     
     
         31 . A kit for detecting single base extension of an oligonucleotide contained in an oligonucleotide array, wherein the kit comprises a first electrode comprising an oligonucleotide array, a mixture of primer extension units, and a polymerase, wherein each different chain-terminating nucleotide species is labeled with a distinguishable electrochemical label capable of participating in a reduction/oxidation reaction at the surface of the first electrode under conditions whereby an electrical potential is applied to the electrode.