JP3902616B2 - Electrochemical detection of nucleic acid hybridization - Google Patents

Description

  The present invention relates to nucleic acid hybridization and sequencing, and more particularly to a method for qualitative and quantitative detection of nucleic acid hybridization and a method for nucleic acid base sequencing.

  Detection of individual DNA sequences in a sample of sudden DNA development provides the basis for a novel approach to gene identification, DNA profiling, and DNA sequencing. One approach to DNA hybridization detection involves the use of surface-bound DNA sequences that can be analyzed using an analytical response that indicates the hybridization of surface-bound oligomers to sequences in a suddenly occurring sample. These analytical methods generally involve laser-induced fluorescence generated from covalently bound labels on the DNA strand of interest that are not sensitive to single base mismatches in the surface-bound duplex. See, for example, US Pat. Nos. 5,143,854 and 5,405,783 to Pirrung et al .; Fodor et al., Nature 364, p. 55 (1993); Bains Angew. Chem. 107, 356 (1995); and Noble's Analytical Chemistry 67 (5), 201A (1995) show the exterior or “chips” for this application. See Hall et al., Biochem. and Molec. Bio. Inter. In an alternative method proposed by Vol. 32 (1), page 21 (1994), DNA hybridization is detected by an electrochemical method involving the observation of redox behavior of single-stranded DNA as a comparison to double-stranded DNA. It was done. This technique is also insensitive to single base mismatches in DNA samples. Techniques for detecting single base mismatch include enzymatic or chemical cleavage studies as shown in US Pat. No. 5,194,372 to Nagai et al. However, these techniques are not useful as long as they require more time and separation techniques.

  US Pat. No. 5,312,527 to Mikkelson et al. Describes a voltammetric sequence-selective sensor for detecting a nucleic acid of interest in which a double-stranded nucleic acid is contacted with a redox active complex. The complex binds nonspecifically to double-stranded DNA. Since the complex itself is a redox-active compound that provides a voltammetric signal, the complex does not function in the analytical method.

  US Pat. No. 4,840,893 to Hill et al. Describes an electrochemical analysis for nucleic acids in which competitive binding behavior between the ligand and the antiligand is sequentially detected electrochemically. .

  Thus, there remains a need in the art for methods of detecting DNA hybridization, including methods of detecting single base pair mismatches that are fast and sensitive and can be quickly applied online.

  In general, the present invention provides a method for detecting a nucleic acid comprising at least one predetermined base (eg, adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine). The method comprises (a) reacting a nucleic acid with a transition metal complex capable of oxidizing a predetermined base by an oxidation-reduction reaction; (b) detecting a redox reaction; (c) a predetermined base. And the step of determining the presence or absence of the nucleic acid from the detected redox reaction. Depending on the particular embodiment of the method and the particular purpose desired, the invention further comprises contacting the nucleic acid with a complementary nucleic acid to form a hybridizing nucleic acid.

  As a first aspect, the present invention provides a method for detecting DNA hybridization. The method comprises (a) contacting a DNA sample with an oligonucleotide probe to form hybridized DNA; (b) the oligonucleotide probe has at least one predetermined base; Reacting with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe by redox reaction; (c) detecting a redox reaction; and (d) detecting at a predetermined base. Determining the presence or absence of hybridizing DNA from the redox reaction. As discussed in detail below, the step of detecting the redox reaction is generally performed by measuring the flow of electrons from a given base.

  In accordance with the second aspect, the present invention provides another method for detecting DNA hybridization. The method includes (a) contacting a DNA sample with an oligonucleotide probe to form hybridized DNA; (b) the oligonucleotide probe has at least one predetermined base, and oxidizes the hybridized DNA. A step of reacting with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe by a reduction reaction; (c) a step of detecting a redox reaction; and (d) a reaction of the detected redox reaction. Measuring the rate; (e) comparing the measured reaction rate with the rate of the redox reaction of the transition metal complex with the single-stranded DNA; and (f) measuring the measured rate of the single-stranded DNA. Determining whether or not the rate of the redox reaction of the transition metal complex is substantially the same.

  In accordance with the third aspect, the present invention provides an apparatus for detecting DNA hybridization. The apparatus includes: (a) a plurality of DNA sample containers; (b) sample handling means for carrying the plurality of DNA sample containers; and (c) an oligonucleotide for delivering an oligonucleotide probe to each of the DNA sample containers. Nucleotide probe delivery means; (d) a transition metal complex delivery means for delivering a transition metal complex to each of the plurality of DNA sample containers; and (e) a redox reaction detector for detecting a redox reaction. including.

  In accordance with the fourth aspect, the present invention provides a second device for detecting DNA hybridization. The apparatus includes: (a) a DNA sample container; (b) an oligonucleotide probe delivery means for delivering a plurality of oligonucleotide probes to the DNA sample container; and (c) a delivery metal complex to the DNA sample container. And (d) a redox reaction detector for detecting a redox reaction.

  According to a fifth aspect, the present invention provides a DNA sequencing method. The method comprises the steps of (a) an oligonucleotide probe comprising a predetermined synthetic base having a unique oxidation potential and contacting a DNA sample with the oligonucleotide probe to form hybrid DNA; The nucleotide probe has a predetermined number of predetermined synthetic bases, and reacts the hybridized DNA with a transition metal complex capable of oxidizing a predetermined synthetic base in the oligonucleotide probe by an oxidation-reduction reaction; c) detecting a redox reaction; (d) measuring a reaction rate of the detected redox reaction; and (e) identifying a base paired with a predetermined synthetic base.

  The foregoing and other aspects of the present invention are explained in detail in the detailed description provided below.

  As used herein, the term “nucleic acid” refers to any nucleic acid containing both DNA and RNA. The nucleic acids of the present invention are typically polynucleotides, a macromolecule of individual nucleotides covalently linked by 3 ', 5' phosphodiester bonds.

  As used herein, “complementary nucleic acid” refers to any nucleic acid, including an oligonucleotide probe that specifically binds to another nucleic acid to create a hybridizing nucleic acid.

  The expression “determining the presence or absence of” qualitatively determines and quantitatively determines the presence or absence of detection behavior (eg, DNA hybridization, RNA hybridization, detection of target nucleic acid, etc.) It is intended to include

  The terms “hybridized DNA” and “hybridized nucleic acid” refer to double-stranded DNA or single-stranded DNA that is hybridized to form a nucleic acid, or triple-helix DNA or nucleic acid. Refers to double-stranded DNA or nucleic acid that is hybridized to form.

  The method and apparatus of the present invention are sometimes described herein with respect to DNA, but this is for purposes of clarity and that the present method and apparatus can be applied to other nucleic acids such as RNA. Should be understood.

A. Nucleic Acid Amplification Methods Since the steps of the present invention involve contacting a DNA sample with an oligonucleotide probe to produce hybridized DNA, certain applications for amplifying DNA prior to contacting the probe are desirable. Amplification of the selected or desired nucleic acid sequence can be performed by any suitable means. In general, D.C. Kwoh and T.W. Kwoh's Am. Biotechnol. Lab. See Volume 8, pages 14-25 (1990). Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction (including reverse transcriptase chain reaction for RNA), ligase chain reaction, strand displacement amplification, transcriptase amplification (D, Kwoh et al. Proc. Natl. Acad Sci. USA 86, 1173-1177 (1989)), self-supporting sequence replication (or “3SR”) (J. Guatelli et al. Proc. Natl. Acad. Sci. USA 87, 1874-1878. (See 1990)), Qβ replicase system (see P. Lizardi et al., Biotechnology Vol. 6, pp. 1197-1220 (1988)), nucleic acid sequence-based amplification (or “NASBA”) (R. Lewis Genetic Engineering News 12 ( 9) Volume 1 (1992), repair chain reaction (or "RCR") (see R. Lewis above), and boomerang DNA amplification (or "BDA") (R. Lewis above). The bases incorporated into the application product are natural or modified bases (improved before amplification or sound) and are selected to optimize the subsequent electrochemical detection step .

  Polymerase chain reaction (PCR) is also performed with known techniques. See, for example, US Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (for examples of all US patents listed herein). The disclosure is to be incorporated by way of example). In general, PCR is performed by first matching each strand of a specific sequence to be detected under hybridization conditions so that an extension product of each primer that is complementary to each strand of nucleic acid is synthesized. One oligonucleotide primer and a nucleic acid sample (eg, in the presence of a thermostable DNA polymerase), where the primer is the other primer when the extension product synthesized from each primer is separated from its complement. Is sufficiently complementary to each strand of the specific sequence to hybridize so that it can act as a template for the synthesis of the extension product of Treating the sample under denaturing conditions to separate the primer extension products from their templates, if present. These steps are repeated periodically until the desired degree of amplification is obtained. Detection of the amplified sequence can be hybridized to the reaction product, and an oligonucleotide probe carrying a detectable label (eg, an oligonucleotide probe of the present invention) is added to the reaction product followed by a known This is done by detecting the label according to the technique. When the nucleic acid to be amplified is RNA, amplification is performed by initial conversion to DNA by reverse transcription according to known techniques.

  Strand displacement amplification (SDA) is performed according to known techniques. In general, G. Walker et al., Proc. Natl. Acad. Sci. USA 89, 392-396 (1992); Walker et al., Nucleic Acids Res. See Volume 20, pages 1691-1696 (1992). For example, SDA is performed using a single amplification primer or a pair of amplification primers, and exponential amplification is achieved with the latter. In general, SDA amplification primers are in the 5 ′ to 3 ′ direction to flanking sequences (non-critical DNA sequences), restriction sites for restriction enzymes used in the reaction, and target sequences to be amplified and / or detected. It consists of an oligonucleotide sequence that hybridizes to (eg, an oligonucleotide probe of the present invention). The flanking sequence, which is arranged to facilitate the binding of the restriction enzyme to the recognition site and provides the DNA polymerase priming site after the restriction site has been nicked, is preferably 15 to 20 nucleotides in length; Functional in the SDA reaction (ie, phosphorothioate linkages bound in the primer strand, as satisfied through the use of non-palindromic recognition sites, do not limit subsequent nicking); The probe is preferably about 13 to 15 nucleotides in length.

  Ligase chain reaction (LCR) is also performed according to known techniques. For example, R.A. See Weiss Science 254, 1292 (1991). In general, one pair binds to one strand of the sequence to be detected and the other pair binds to the other strand of the sequence to be detected: performed with two pairs of oligonucleotide probes. Each pair is completely overlapped by the corresponding strand. The reaction initially denatures (eg, separates) the strands of the sequence to be detected, and then couples the strands in the presence of a thermostable ligase so that each pair of oligonucleotide probes is bound together. It reacts with the nucleotide probe and then the reaction products are separated, after which the process is repeated periodically until the sequence is amplified to the desired extent. Detection is then performed in a manner similar to that described above for PCR.

B. Oligonucleotide Probe As described above, the production method of the present invention is useful for detection of DNA hybridization. The first step of the production method involves contacting the DNA sample with an oligonucleotide probe to form hybridized DNA. Oligonucleotide probes useful in the methods of the invention consist of between about 4 or 6 bases and about 80 or 100 bases or more, more preferably between about 8 and about 15 bases. The probe may be Oligonucleotide probes are prepared with any of a wide variety of base sequences according to techniques well known in the art. Suitable bases for the preparation of oligonucleotide probes include naturally occurring nucleotide bases such as adenosine, cytosine, guanine, uracil, and thymine; and 8-oxo-guanine, 6-mercaptoguanine, 4-acetylcytidine, 5- ( Carboxyhydroxyethyl) uridine, 2′-O-methylcytidine, 5-carboxymethylamino-methyl-2-thiolysine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2′-O-methylpsuduridine, β, D -Galactosylkaeosin, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2- Tilguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, β, D-mannosylkeosin, 5- Methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl) carbamoyl) threonine, N-((9 -Β-D-ribofuranosylpurin-6-yl) N-methyl-carbamoyl) threonine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, wybutoxosine, psuduridine, keosin , 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 2-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl) carbamoyl) threonine, 2 ′ Non-naturally occurring or “synthetic” nucleotide bases such as —O-methyl-5-methyluridine, 2′-O-methyluridine, wybutosine, and 3- (3-amino-3-carboxypropyl) uridine Selected from. The backbone of the oligonucleotide is used including DNA, RNA (although RNA is less preferred than DNA), modified sugars such as carbocycles, and sugars containing 2 ′ substitutions such as fluoro and methoxy. Oligonucleotides have at least one or all internucleotide linked phosphate residues such as methylphosphonate, methylphosphonothioate, phosphoromorpholidate, phosphoropiperazidate and phosphoramidate (eg, every other Oligonucleotides which are improved phosphates of internucleotide-linked phosphate residues (modified as described). The oligonucleotides are P.I. A “peptide nucleic acid” as described in Nielsen et al., Science 254, pages 1497-1500 (1991). The oligonucleotide probe need only retain at least a portion that can bind at least a known portion of the sequence of the DNA sample. It can be used in some applications to combine a DNA sample with a number of oligonucleotide probes having different base sequences (eg, when two or more nucleic acids of interest are present in a sample, or a single nucleic acid of interest is a “sandwich” Preferred when hybridized to two or more probes).

C. Hybridization Methodology A DNA (or nucleic acid) sample is contacted with an oligonucleotide probe in any suitable manner known to those skilled in the art. For example, a DNA sample is dissolved in a solution and contacted with an oligonucleotide probe by dissolving the oligonucleotide probe in a solution containing the DNA sample under conditions that allow hybridization. Appropriate conditions are known to those skilled in the art (see, eg, US Pat. No. 4,358,535 to Falkow et al. And other US patent examples that describe the same), including high salt concentration conditions. . Alternatively, the DNA sample is dissolved in a solution with oligonucleotide probes immobilized on a solid support, and the DNA sample is immersed in a solution containing the DNA sample with the solid support having oligonucleotide probes immobilized thereon. In contact with the oligonucleotide probe.

D. Oxidizing agent and redox reaction When the hybridization stage proceeds to the oxidation stage, after hybridization, the hybridization DNA (or nucleic acid) is capable of oxidizing a given base in the oligonucleotide probe in an oxidation-reduction reaction. Reacted with oxidant. A given base can be a naturally occurring or synthetic nucleotide base in an oligonucleotide probe that undergoes oxidation in reaction with a selected oxidizing agent. A given base exhibits a unique oxidation rate when paired as compared to when a given base is not paired. A given base exhibits a unique oxidation rate when paired with each of the four naturally occurring bases. In general, bases of 5′-mononucleotides (eg, 5′-deoxyribonucleotides or 5′-ribonucleotides) exhibit rate constants greater than 10 ⁴ M ⁻¹ s ⁻¹ that can be detected using catalytic reactions. Examples of suitable predetermined bases include, but are not limited to, guanine, adenosine, 8-oxo-guanine, and 8-oxo-adenosine, 8-bromo-guanine, guanosine, xanthosine, wiosine, pseudouridine, 6-mercaptoguanine, 8-mercaptoguanine, 2-thioxanthine, 6-thioxanthine, 6-mercaptopurine, 2-amino-6-carboxymethyl-mercaptopurine, 2-mercaptopurine, 6-methoxypurine, 2-acetylamino-6-hydroxy Purine, 6-methylthio-2-hydroxypurine, 2-dimethylamino-6-hydroxypurine, 2-hydroxypurine, 2-aminopurine, 6-amino-2-dimethylallyl-purine, 2-thioadenine, 8-hydroxyadenine , Containing 8-methoxyadenine Typically, the predetermined base is selected from the group consisting of guanine, adenosine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine, where guanine is a presently preferred naturally occurring predetermined base. Yes, 6-mercaptoguanine is the currently preferred synthetic base.

An oxidant is a molecule having a charge, such as a cationic, anionic, or Zwitter ionic molecule that is reactive with a given base at a unique oxidation potential. Thus, the choice of oxidant can be quickly determined by one skilled in the art according to the specific predetermined base selected. Particularly preferred oxidizing agents include transition metal complexes capable of metal-DNA electron transfer with a given base such that the catalytic cycle is completed and the reduced form of the metal complex is regenerated. Examples of suitable transition metal complexes for the process according to the invention are, for example, ruthenium ²⁺ (2,2′-bipyridine) ₃ (“Ru (bpy) ₃ ²⁺ ”), ruthenium ²⁺ (4,4′-dimethyl-2, 2′-bipyridine) ₃ (“Ru (Me ₂ -bpy) ₃ ²⁺ ”), ruthenium ²⁺ (5,6-dimethyl-1,10-phenanthroline) ₃ (“Ru (Me ₂ -phen) ₃ ²⁺ ”), Iron ²⁺ (2,2′-bipyridine) ₃ (“Fe (bpy) ₃ ²⁺ ”), iron ²⁺ (5-chlorophenanthroline) ₃ (“Fe (5-Cl-phen) ₃ ²⁺ ”), osmium ²⁺ (2 , ^{_{2'-bipyridine) 3 ( "Os (bpy)}} 3 2+"), osmium ²⁺ _{(5-chloro-phenanthroline) 3 ( "Os (5-} Cl-phen) 3 2+"), Jiokisoruteniu ¹⁺ phosphine, and a di-oxo ruthenium ¹⁺ pyridine ^{_{( "ReO 2 (py) 4}} 1+"). Anionic complexes useful as oxidizing agents are: Ru (bpy) ((SO ₃ ) ₂ -bpy) ₂ ²⁻ and Ru (bpy) ((CO ₂ ) ₂ -bpy) ₂ ²⁻ , useful as oxidizing agents The Zwitter ionic complex is (SO ₃ ) ₂ -bpy ^2- is 4,4'-disulfonato-2,2'-bipyridine and (CO ₂ ) ₂ -bpy ² is 4,4'-di. Carboxy-2,2′-bipyridine, Ru (bpy) ₂ ((SO ₃ ) ₂ -bpy) and Ru (bpy) ₂ ((CO ₂ ) ₂ -bpy). Suitable substituted derivatives of pyridine, bipyridine and phenanthroline are also used in complexes with any of the aforementioned metals. Suitable substituted derivatives include, but are not limited to, 4-aminopyridine, 4-dimethylpyridine, 4-acetylpyridine, 4-nitropyridine, 4,4′-diamino-2,2′-bipyridine, 5,5′- Diamino-2,2′-bipyridine, 6,6′-diamino-2,2′-bipyridine, 4,4′-diethylenediamine-2,2′-bipyridine, 5,5′-diethylenediamine-2,2 ′ -Bipyridine, 6,6'-diethylenediamine-2,2'-bipyridine, 4,4'-dihydroxyl-2,2'-bipyridine, 5,5'-dihydroxyl-2,2'-bipyridine, 6, 6'-dihydroxyl-2,2'-bipyridine, 4,4 ', 4 "-triamino-2,2', 2" -terpyridine, 4,4 ', 4 "-triethylenediamine-2,2', 2 "-Terpyridine, 4,4 ', 4" -Trich Roxy-2,2 ', 2 "-terpyridine, 4,4', 4" -trinitro-2,2 ', 2 "-terpyridine, 4,4', 4" -triphenyl-2,2 ', 2 " -Terpyridine, 4,7-diamino-1,10-phenanthroline, 3,8-diamino-1,10-phenanthroline, 4,7-diethylenediamine-1,10-phenanthroline, 3,8-diethylenediamine-1,10 -Phenanthroline, 4,7-dihydroxy-1,10-phenanthroline, 3,8-dihydroxy-1,10-phenanthroline, 4,7-dinitro-1,10-phenanthroline, 3,8-dinitro-1,10-phenanthroline 4,7-diphenyl-1,10-phenanthroline, 3,8-diphenyl-1,10-phenanthroline, 4,7-disperamine-1 10-phenanthroline, 3,8-Jisuperamin-1,10-phenanthroline, and dipyrido [3,2-a: 2 ', 2'-c] containing phenazine.

  The oxidant is reacted with the hybridizing DNA according to a suitable technique to achieve an oxidant redox reaction with a given base. All that is required is that the oxidant is reacted with the hybridizing DNA under conditions effective to achieve selective oxidation of a given base. For example, transition metals are dissolved by dissolving the oxidant in a solution containing dissolved hybridized DNA under conditions sufficient to cause a redox reaction between the oxidant and a given base. Reacted with the hybridized DNA. Alternatively, in an embodiment in which DNA is immobilized on a solid support, an oxidant is immobilized on the same solid support, and the solid support undergoes a redox reaction between the oxidant and a predetermined base. The oxidant is reacted with the hybridizing DNA by immersion in the solution under sufficient conditions. The solvent in which the redox reaction occurs is a suitable solvent for dissolving DNA, preferably water. Appropriate conditions for causing the redox reaction are known to those skilled in the art.

In hybridizing DNA or nucleic acids, the oxidizing agent binds in a small groove in the DNA, and intimate contact between a given base and the oxidizing agent is hindered by the unique structure of the double (or triple) helix. This defense of a given base residue results in the need for electron tunneling through a solvent that reduces the rate of electron transfer. The accessibility of the solvent varies with the nature of the nucleotide base paired with a given base. The tunneling distance is the formula:
k / k _ss = exp (−βΔr)
Where Δr is the change in distance in the duplex compared to the single strand and k _ss is the rate constant for oxidation of a given base in the single-stranded DNA sample. That is, the tunneling distance between a given base and oxidant is different for each base pairing and for unpaired DNA. Thus, the electron transfer rate constant indicates the identity of the paired (or incorrectly paired) bases. Given that the driving force for electron transfer is not greater than the rearrangement energy (λ), the plot of RTlnk against driving force, corrected for the work term coupled with the approach of the reactants, is ½ slope according to Marcus theory Form a straight line with Based on Marcus theory, the absolute rate constant is:
k = νexp [−β (r−r ₀ )] exp [− (ΔG + λ) ² / 4λRT]
Where ν is the rate constant in diffusion rate (10 ¹¹ M ⁻¹ s ⁻¹ ), r is the distance between the reactant and product in the activated complex, and r ₀ is the closest distance between the reactant and product, and β is the effect of the intervening medium. As mentioned above, since a given base is incorporated within the hybridizing DNA, this imposes a finite distance that the electrons must tunnel to the oxidant. In other words, r is not equal to _{r 0.} Β for water is about 3A- ¹ . This relatively large value for β indicates that a large change in the electron transfer rate constant will be caused by a very small change in the tunneling distance. The conformation of DNA between a given base and a base paired with a given base depends on the base paired with the given base, and the base paired with the given base has an electron This affects the tunneling distance that must be tunneled between the base and the oxidant. A correlation between the tunneling distance and a particular base paired with a given base is thus established.

E. Detection of redox reaction Occurrence of the redox reaction is detected according to suitable means known to those skilled in the art. For example, the occurrence of a redox reaction is detected using a detection electrode for observing a change in an electrical signal indicative of the occurrence of a redox reaction. Typically, a detection electrode that is sensitive to electron transfer between the oxidizing agent and the hybridizing DNA is placed in contact with a solution containing the reactive hybridizing DNA and the oxidizing agent. In general, a reference electrode and an auxiliary electrode are also placed in contact with the solution along with the detection electrode (most current passes through the auxiliary electrode). Suitable detection electrodes are well known to those skilled in the art and include, for example, glassy carbon electrodes or indium tin oxide electrodes. Similarly, suitable reference electrodes are also well known to those skilled in the art and include, for example, silver / silver chloride electrodes.

  Detection of the electrical signal associated with the redox reaction allows determination of the presence or absence of hybridizing DNA. The step of determining the presence or absence of hybridization DNA typically comprises (i) measuring the reaction rate of the redox reaction, and (ii) determining the measured reaction rate from the transition metal with the single-stranded DNA. Comparing with the redox reaction rate of the complex; and (iii) determining whether the measured reaction rate is substantially the same as the redox reaction rate of the transition metal complex with the single-stranded DNA. Including. The step of measuring the reaction rate is carried out by any suitable means. For example, the relative reaction rate can be determined by comparing the current as a function of sweep rate, probe concentration, target concentration, vehicle, buffer, temperature, and / or electrochemical method.

  The redox reaction rate is measured according to suitable means known to those skilled in the art. Typically, the redox reaction rate is measured by measuring an electrical signal coupled to the occurrence of the redox reaction. For example, the electrical signal coupled to the redox reaction is measured by providing a suitable device in electrical communication with the sensing electrode. A suitable device can measure the electrical signal generated to provide a redox kinetic measurement of the reaction between the hybridizing DNA and the oxidizing agent. The electrical signal is specific to any electrochemical method including cyclic voltammetry, normal pulse voltammetry, chronoamperometry, and square wave voltammetry, which is a recently preferred form .

  The measured reaction rate is then compared to the known redox reaction rate of the transition metal complex with single stranded DNA. As already discussed in detail, in both hybridization and single-stranded DNA, the tunneling distance between the oxidant and the given base is the redox reaction of the reaction between the oxidant and the given base. Affects speed. Accordingly, the hybridized DNA exhibits a different redox reaction rate than single-stranded DNA. Presence or absence of hybridization DNA at a given base is based on whether the measured redox reaction rate is the same as the redox reaction rate between the oxidizing agent and the given base in the single-stranded DNA. Can be determined. Furthermore, the tunneling distance between the oxidant and a given base varies according to the binding distance between a given base and its counterpart such that each possible base pairing is distinguished from the others. The bond distance between a given base and its base pair depends on the base paired with the given base. For example, the redox rate for the oxidation of guanine paired with adenosine is different from the redox rate for the oxidation of guanine paired with cytosine, which in turn has a redox rate for the oxidation of guanine paired with cytosine. The redox rate for the oxidation of guanine paired with guanine and the redox rate for the oxidation of guanine paired with guanine differ from the redox rate for the oxidation of guanine paired with thymine. In particular, the redox rate for oxidation of guanine is greater than guanine paired with adenosine for single-stranded guanine, larger than guanine paired with guanine, and paired with guanine. It follows the trend that guanine is larger than guanine paired with thymine and larger than guanine paired with guanine cytosine paired with thymine. Therefore, the method of the present invention is useful for detecting an incorrect pairing of a single base pair at a predetermined base or a base pair adjacent to the predetermined base.

  Advantageously, the difference between the redox kinetics of oxidation of a given base when paired with each of a variety of naturally occurring bases also allows the identification of the base paired with a given base. To do. A base paired with a given base (i) measures the reaction rate of the detected redox reaction and (ii) measures the measured reaction rate with adenosine, cytosine, guanine, or By comparing each of the four different known redox kinetics of the thymine-containing DNA and the oxidant, and (iii) by determining which of the known kinetics is substantially the same as the measured kinetics. Identified. The reaction rate is measured according to the technique described above. Similarly, the kinetics of each of four different redox reactions of DNA and oxidant with adenosine, cytosine, guanine, or thymine bound to a given base is the same technique as these kinetics are known. Measured according to The measured reaction rate of the redox reaction of the oxidant with the hybridizing DNA is then known as the known redox reaction rate of the DNA and oxidant with adenosine, cytosine, guanine, or thymine bound to a given base. To be compared. For example, the base paired with a given base is determined by determining a known base pairing having a redox reaction rate that is substantially the same as the measured redox reaction rate.

F. DNA Sequencing The present invention comprises the steps of (a) contacting a DNA sample with an oligonucleotide probe to form a hybridized DNA, wherein the oligonucleotide probe comprises a predetermined synthetic base having a unique oxidation potential; ) The oligonucleotide probe has a predetermined number of predetermined synthetic bases, and the hybridized DNA is reacted with an oxidant such as a transition metal complex capable of oxidizing the predetermined synthetic base in the oligonucleotide probe by an oxidation-reduction reaction. (C) detecting a redox reaction; (d) measuring a reaction rate of the detected redox reaction; and (e) identifying a base paired with a predetermined synthetic base. A method for sequencing DNA comprising the steps is also provided.

  According to the methods discussed so far, the DNA sample is amplified prior to contacting the oligonucleotide probe according to techniques known to those skilled in the art. The synthetic base is selected from the group of bases described so far and other synthetic bases known to those skilled in the art. The only restriction is that the synthetic base must have a unique oxidation potential compared to that of four naturally occurring bases, namely adenosine, cytosine, guanine, and thymine. The step of reacting the hybridized DNA with an oxidizing agent, detecting the redox reaction, and contacting the DNA sample with an oligonucleotide probe that measures the reaction rate is performed as described above. The step of identifying a base paired with a predetermined synthetic base consists of (i) measuring the reaction rate with a DNA having adenosine, cytosine, guanine, or thymine bound to the predetermined synthetic base and an oxidizing agent 4. Comparing to each of two different known redox reaction rates, and (ii) determining substantially the same reaction rate measured among the known reaction rates.

  In another example, the oligonucleotide probe further comprises a second predetermined synthetic base. The second predetermined synthetic base has a unique oxidation potential that is different from the oxidation potential of the first predetermined synthetic base. In this embodiment, the step of detecting the redox reaction between the predetermined base and the oxidizing agent further includes the step of detecting the redox reaction between the same second predetermined synthetic base and the oxidizing agent. In addition, the step of measuring the redox reaction rate includes measuring the redox reaction rate of the second predetermined base and oxidation with the same oxidizing agent. Further, the step of identifying a base paired with a predetermined synthetic base further comprises the step of identifying a base paired with a similar second predetermined synthetic base. According to this example, redox reactions of both predetermined bases are detected, and eventually the base paired with each predetermined synthetic base is identified using the method described so far. As will be apparent to those skilled in the art, each given synthetic base is distinct from the oxidation potential of all other given synthetic bases and exhibits a unique oxidation potential that is different from the oxidation potential of each of the four naturally occurring bases. In some cases, the method is carried out using two or more predetermined synthetic bases. Not only is each base paired with a given base identified according to the method described here, but the DNA has a sufficient number of given synthetic bases at different sites to identify each base in the DNA sample. Sequencing is carried out by repeating the steps of the above method using different oligonucleotide probes. In other words, the DNA sample contains a sufficient number of each probe sequence containing at least one predetermined synthetic base, which differs along the probe sequence in each oligonucleotide probe and is placed at the calculated site. Sequencing is provided by providing an oligonucleotide probe. In this method, detection of repeated redox reaction of hybrid DNA with an oxidizing agent, measurement of redox reaction rate, and identification of a base paired with a predetermined synthetic base are performed as base-to-base identification of a DNA sample sequence. Bring.

G. Apparatus The present invention also provides an apparatus useful for performing the method of the present invention. One such illustrative device is schematically illustrated in FIG. In general, the apparatus comprises a number of DNA sample containers 10. The drive assembly 11 is arranged as a sample handling means for carrying a number of DNA sample containers. The liquid reservoir 12, the supply line 13 and the valve 14 are arranged as oligonucleotide probe delivery means for delivering the oligonucleotide probe to each DNA sample container, and the corresponding liquid reservoir 15, the supply line 16 and the valve 17 are arranged in a large number. It is arranged as an oxidant delivery means for delivering the transition metal complex to each of the DNA sample containers. The probe assembly 20 including the driving device 21 and the probe 22 is arranged as a redox reaction detecting means for detecting a redox reaction. During operation, the DNA sample is pre-deposited in the sample container 10. The drive assembly 11 then transports the sample container 10 continuously below the oligonucleotide probe delivery means and the oxidant delivery means for delivering the respective reagents therein. After delivery of the reagent, each sample container is advanced to a position below the probe 22 by the driving means, and the probe 22 is advanced into the sample container by the driving device 21 for detecting the oxidation-reduction reaction. Additional electrodes necessary for implementation in the cyclic voltammogram are carried together with the probe 22. Various component operations and data collection are performed using a suitable controller 30, such as a software program running on a general purpose computer.

  Many variations of the device are of course apparent to those skilled in the art. A number of DNA sample containers are suitable containers known to those skilled in the art and include microtiter plates, test tubes, petri dishes, culture vials, solid supports, etc. that can contain DNA samples. The sample handling means is a suitably designed sample container handling means known to those skilled in the art capable of carrying a DNA sample container.

  Suitable oligonucleotide probe delivery means for delivering oligonucleotide probes to each DNA sample container are well known in the art. For example, according to one embodiment, the oligonucleotide probe delivery means comprises a solid support on which the oligonucleotide probe is immobilized. This oligonucleotide probe delivery means needs to allow sufficient contact between the DNA sample and the oligonucleotide probe under appropriate conditions that result in hybridization of the DNA sample and the oligonucleotide probe. Suitable oxidant delivery means for delivering oxidant to each of a number of DNA sample containers are well known in the art. For example, according to one embodiment, the oxidant is attached to a solid support comprising oxidant delivery means. A redox reaction detector for detecting a redox reaction comprises one or more electrodes capable of detecting oxidation of a predetermined base according to one embodiment. Suitable detection and reference electrodes have been described so far with examples to the method of the present invention. Preferably, the electrode is in electrical communication using means for measuring the redox reaction rate of the redox reaction. Suitable means for measuring the oxidation-reduction reaction rate are known to those skilled in the art as described above.

  In another embodiment of the apparatus of the present invention, an apparatus for detecting DNA hybridization comprises: (a) a DNA sample container; (b) an oligonucleotide probe delivery means for delivering a plurality of oligonucleotide probes to the DNA sample container; (C) an oxidant delivery means for delivering an oxidant to the DNA sample container; and (d) a redox reaction detector for detecting the redox reaction. US Pat. Nos. 5,143,854 and 5,405,783 to Pirrung et al .; Fodor et al., Nature 364, page 555 (1993), the disclosure of which is hereby fully incorporated herein by reference. Years); Bains Angew. Chem. 107, 356 (1995); and Noble, Analytical Chemistry 67 (5), 21 (1995), etc., and is adapted for use with fixed probes.

  As noted above, the DNA sample container is a suitable container known to those skilled in the art. The oligonucleotide probe delivery means is preferably a solid support having a number of oligonucleotide probes immobilized thereon, which can deliver the probe to a DNA sample container. For example, according to one embodiment, a solid support having a number of oligonucleotide probes immobilized thereon has sufficient conditions to allow hybridization with one or more oligonucleotide probes of the DNA sample. A DNA sample is contacted with the DNA sample in the bottom.

  Appropriate oxidant delivery means for delivering oxidant to the DNA sample container have been described so far. A preferred oxidant delivery means comprises a solid support having an oxidant immobilized thereon. According to one embodiment, the oxidant and multiple oligonucleotide probes are immobilized on the same solid support.

  The apparatus according to the present invention is useful for performing diagnostic analysis of changes in DNA samples. A large number of oligonucleotide probes allow analysis and detection of various DNAs within a single sample, thus providing a useful tool for screening a single sample for DNA changes, including pathogens and viruses.

H. RNA Hybridization Detection, RNA Sequencing and RNA Fraud Pairing Detection Also disclosed herein are RNA hybridization detection methods, RNA sequencing methods and RNA fraud pair detection methods. RNA useful for performing such methods includes, but is not limited to, ribosomal RNA, transfer RNA, or pathogenic RNA (eg, RNA obtained from RNA viruses such as retroviruses, HIV-1 etc.). The first aspect of the present invention thus comprises (a) contacting an RNA sample with an oligonucleotide probe to form hybridized RNA; (b) the oligonucleotide probe having at least one predetermined base. Reacting the hybridized RNA with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe by a redox reaction; (c) detecting a redox reaction; (d) A method of detecting hybrid RNA comprising the step of determining the presence or absence of hybrid RNA from the detected redox reaction at a predetermined base.

  More specifically, a method of detecting hybridized RNA includes (a) contacting an RNA sample with an oligonucleotide probe to form hybridized RNA; (b) the oligonucleotide probe comprises at least one predetermined base. And reacting the hybridized RNA with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe by an oxidation-reduction reaction, and (c) detecting the oxidation-reduction reaction; (E) measuring the reaction rate of the detected redox reaction, (e) comparing the measured reaction rate with the oxidation-reduction reaction rate of the transition metal complex with the single-stranded RNA, and (f) measuring. Determining whether or not the reaction rate obtained is substantially the same as the rate of the redox reaction of the transition metal complex with the single-stranded RNA.

  The method of RNA sequencing comprises the steps of (a) contacting an RNA sample with an oligonucleotide probe to form a hybridized RNA, wherein the oligonucleotide probe comprises a predetermined base having a unique oxidation rate; The oligonucleotide probe has a predetermined number of predetermined bases, and reacts the hybridized RNA with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe by an oxidation-reduction reaction; (c) It comprises a step of detecting a redox reaction, (d) a step of measuring the reaction rate of the detected redox reaction, and (e) a step of identifying a base paired with a predetermined synthetic base. Oligonucleotide probes, hybridization methodologies, oxidants, redox reaction detection, and devices useful for performing these methods are substantially shown in Sections AH above and are known to those skilled in the art. It applies to the use of RNA as a nucleic acid sample according to the principle (eg replacing uracil with the base thymine).

I. Detection of a given base on a nucleic acid of interest In a method already described in particular, a metal complex is used to obtain an electrochemical current from single- and double-stranded DNA or nucleic acid. Certain bases such as guanine give an electrical signal, which is much weaker than double-stranded DNA. Such a method effectively demonstrates high structural sensitivity and can identify single base mismatches. Such a method is therefore particularly useful for DNA sequencing. However, two disadvantages of such a method are: (a) there is a negative signal in going from the probe strand to the hybrid, and (b) no signal amplification. The following techniques provide solutions to these problems. In addition, the following techniques are particularly useful for diagnostic analysis and are particularly useful for quantitative detection of nucleic acids.

  In view of the above, what is also disclosed herein is a method for detecting the presence and absence of a target nucleic acid in a test sample presumed to contain at least one predetermined base and the same nucleic acid. is there. In contrast to the above method, in the present invention, the predetermined base is placed on the target nucleic acid rather than on the oligonucleotide probe.

  The method is performed on a test sample containing the nucleic acid of interest. Any presumed to contain nucleic acid of interest, including (but not limited to) tissue samples such as biopsy samples and biological fluids such as blood, sputum, urine and semen samples, bacterial cultures, soil samples, food samples etc. These test samples are used. The nucleic acid of interest is of any origin, including animals, plants, or microorganisms (eg, viruses, procariotic and eucaliotic bacteria, protozoa, fungi, protoctistas, etc.), depending on the specific purpose of the test. The sample is processed or purified according to well-known techniques known to those skilled in the art prior to performing the method; if desired, the nucleic acid is digested, disrupted and / or amplified (if desired) before the method is performed. See above).

  As schematically shown in FIG. 4, the method comprises: (a) contacting a test sample with an oligonucleotide probe that specifically binds to a nucleic acid of interest to form a hybridizing nucleic acid; and (b) hybridization. Contacting the nucleic acid with a transition metal complex that oxidizes a given base in a redox reaction; (c) detecting the presence or absence of a redox reaction associated with the hybridizing nucleic acid; and (d) a given The step of determining the presence or absence of the target nucleic acid in the test sample from the redox reaction detected with the base. As shown in FIG. 4, the oligonucleotide probe involves a separation step that occurs before the detection step (eg, between steps (a) and (b) or between steps (b) and (c)), Immobilized on a solid support to facilitate separation of the test sample from the hybridizing nucleic acid. In addition, oligonucleotide probes can be obtained in solution and (for example, by a mediator that binds to the oligonucleotide probe or by biotin-avidin binding interactions in which biotin is bound to the oligonucleotide probe and avidin is immobilized on a solid support. ) Freely provided among other means provided for the separation of hybridizing nucleic acids from the sample.

  Preferably, the nucleic acid of interest comprises at least 10 or more more predetermined bases than the oligonucleotide probe comprises, or more preferably comprises at least 50 or 100 or more more predetermined bases than the oligonucleotide probe comprises. When the nucleic acid of interest contains more predetermined bases than oligonucleotide probes, a greater current increase is beneficially obtained.

  In addition, the oligonucleotide probe is free of a given base, or at least substantially free of a given base (ie, the signal from the probe does not interfere or is not mistaken for the signal from the target nucleic acid) Containing a small number of predetermined bases). If a sequence of naturally occurring bases that readily hybridize to the nucleic acid of interest is not available, another base utilization plan that is redox inactive (discussed below) is used.

  As shown in FIG. 4, the nucleic acid of interest is preferably longer than the oligonucleotide probe, and at least one predetermined base is not hybridized to the oligonucleotide probe in the hybridizing nucleic acid (ie, is a “protruding” base). Preferably at least 10, 50, or 100 predetermined bases are “protruding” bases that provide substantial amplification of the electrochemical signal detected thereby.

For example, oligonucleotide probes that do not contain any guanine residues (eg, only A, T, and C) are used. The cyclic voltammogram of Ru (pby) ₃ ²⁺ in the presence of this chain is very similar to that without oligomers. This strand then contains guanine in either or both of the overlapping base pair region and the overhang region, assuming that the nucleic acid of interest is longer than the oligonucleotide probe (indicated by “G” adjacent to the strand of nucleic acid of interest in FIG. ) Hybridized with the strand of interest. Since a large number of guanines are detected, the signal is amplified compared to the number of hybrids. When genomic DNA or RNA is the strand of interest, a number of overhanging guanines are encountered that give large signal amplification. For example, ribosomal RNA contains as many as 1000 guanines for a particular organism, thus providing approximately 1000-fold amplification per hybridization result. For example, in one preferred embodiment, the analysis for a given base on the strand of interest provides a low background signal when swept in the presence of a mediator, a solid surface oriented near the electrode surface Includes immobilization of the above (preferably non-redox) probe strands. The solid surface is then contacted with a solution of the desired chain containing a given base. If hybridization occurs, the chain of interest is now in the immediate vicinity of the electrode and an increase in current is detected.

Nucleic acid quantification. The present invention is particularly well suited for quantitative detection of nucleic acids. In the case described in this section, the rate constant for hybrid oxidation by an oxidant (eg, Ru (pby) ₃ ²⁺ ) can be determined from a cyclic voltammogram (or other electrical signal) by digital simulation. Under most conditions, the reaction follows a second order kinetics, ie rate = k [Ru (bpy) ₃ ²⁺ ] [DNA], where k is specific for a particular probe-object hybrid. Rate constant, [Ru (bpy) ₃ ²⁺ ] is the concentration of oxidizing agent, and [DNA] is the concentration of hybrid (which can be a DNA-RNA hybrid). If k and [Ru (bpy) ₃ ²⁺ ] are known, the amount of hybrid can be determined. In fact, a calibration curve was constructed for the current increase obtained with different amounts of standard solution containing the target DNA and RNA, and the current increase was used to directly obtain the amount of hybrid. This amount is then directly compared with the amount of the target substance. For example, M.M. Houdniy et al. Virol. 69, 3510-3516 (1995); See Mellors et al., Science 272, 1167-1170 (1996).

  Oligonucleotide probes, hybridization methodologies, oxidizing agents and methods of redox reactions, detection of redox reactions, and equipment useful for performing these methods are as shown in Sections AH above.

J. et al. Redox Inactive Substitute Base One disadvantage of the method described in Section H above is that the oligonucleotide probe preferably does not contain a substantial number of predetermined bases (eg, guanine). The solution to this problem is to replace guanine in the probe strand (ie, a base that has a higher binding affinity than other bases show in the nucleic acid duplex, such as guanine) and the appropriate reaction The use of surrogate bases that are not oxidized by the oxidant under conditions. Examples of such surrogate bases where guanine is the predetermined base are inosine and 7-deaza-guanine.

  As a result, when the target nucleic acid contains at least one predetermined base and the probe or capture nucleic acid contains a substitute redox-inactive base, the method for detecting the target nucleic acid (a) Contacting the nucleic acid of interest with a complementary nucleic acid that specifically binds to the nucleic acid; (b) contacting the hybridizing nucleic acid with a transition metal complex capable of oxidizing a given base in a redox reaction; And (d) determining the presence or absence of the nucleic acid from the detected redox reaction at a predetermined base. If the predetermined base in the target nucleic acid is guanine and the target nucleic acid contains cytosine (which normally binds to guanine in the complementary nucleic acid), the complementary nucleic acid contains a surrogate base that binds to cytosine in the hybridizing nucleic acid. The surrogate base is selected from the group consisting of inosine and 7-deaza-guanine. The reaction step typically consists of reacting the nucleic acid with the transition metal complex under conditions sufficient to achieve selective oxidation of a given base without oxidizing the surrogate base.

  Oligonucleotide probes, hybridization methodologies, oxidants and redox reaction methods, redox reaction detection, and equipment useful for performing these methods are as described in Sections A-I above.

K. Polymerization of a given base using terminal transferase Another embodiment of the method described in Section H above includes extending a nucleic acid of interest using a terminal transferase to provide an additional version of the given base. . As shown in FIG. 7, such a method comprises: (a) the oligonucleotide probe has a termination that is blocked for extension by terminal transferase and specifically binds to the nucleic acid of interest to form a hybridizing nucleic acid. Contacting the test sample with the oligonucleotide probe; and (b) placing the oligonucleotide probe in a solution containing a predetermined base in the presence of a terminal transferase to produce an extension product of the target nucleic acid consisting of the predetermined base. (C) contacting the oligonucleotide probe with a transition metal complex that oxidizes a given base in a redox reaction; (d) detecting the presence or absence of the redox reaction; e) Presence or absence of the target nucleic acid in the test sample from the detected redox reaction at a predetermined base Consisting of Mel stage. The test sample is preferably separated from the oligonucleotide probe prior to the detection step, more preferably from the probe between steps (a) and (b) above. Separation is performed by use of a fixed probe, or the probe is freely provided in solution as described in section H above.

  Oligonucleotide probes, hybridization methodologies, oxidants and redox reaction methods, redox reaction detection, and equipment useful for performing these methods are as described in Sections A-I above.

L. Sandwich Analysis Another example of the method of section H above is the so-called “sandwich” analysis schematically shown in FIG. In sandwich analysis, the nucleic acid of interest is part of a three (or more) part hybrid consisting of a capture probe, a nucleic acid of interest, and a signal probe.

  A method for detecting the presence or absence of a nucleic acid of interest in a test sample presumed to contain the same comprises: (a) the capture probe specifically binds to the nucleic acid of interest and provides an oligonucleotide capture probe (B) contacting the test sample with a capture probe to form a hybridizing nucleic acid; (c) a signal probe that binds specifically to the nucleic acid of interest and produces a hybridizing nucleic acid sandwich. The signal probe comprises at least one predetermined base and contacting the oligonucleotide signal probe with the hybridizing nucleic acid; and (d) oxidizing the hybridized nucleic acid sandwich with the redox reaction of the predetermined base. (E) the presence of a redox reaction associated with the hybridizing nucleic acid; Or step and detecting an absence; consisting of step (f) determining the presence or absence of the detected nucleic acid of interest in a test sample from the oxidation-reduction reaction at a given base. The separation step is between step (b) and step (c) or between step (c) and step (d), and the test sample is preferably separated from the capture probe. Depending on the analytical format (eg, sudden or homogenous), the oligonucleotide capture probe is hybridized from a solid support (eg, the inner surface of a polymer bead, plate, or microtiter plate well), or from a test sample as described above. Immobilized on an alternative means provided to separate the nucleic acids.

  A number of “sandwich” analysis formats are known. The choice of analysis format is not critical and an appropriate format is used to practice the invention. For example, oligonucleotide capture probes are immobilized on a solid support, as described in US Pat. No. 4,486,539 to Ranki et al. As described in U.S. Pat. No. 4,868,104 to Kurn et al., An oligonucleotide probe contains a polymer-forming unit, and the polymerisation separates the hybridized nucleic acid sandwich. M.M. S. Urdea's Clinical Chem. As described in 39, pages 725-726 (1993), the signal probe is linear or branched. Mediator polynucleotides that bind oligonucleotide capture probes to immobilized polynucleotides are used as described in US Pat. No. 4,751,177 to Stabinsky. R. Goodson European Patent Application No. 0 238 332: Harrison European Patent Application No. 0 139 489, and N.E. As described in Dattagupta European Patent Application No. 0 192 168, oligonucleotide probes are part of a specific binding pair (eg, biotin) and immobilized on a solid support (eg, avidin), binding The hybridized nucleic acid sandwich is separated from the test sample by a second binding interaction with the other part of the pair.

  Oligonucleotide probes, hybridization methodologies, oxidants and redox reaction methods, redox reaction detection, and equipment useful for performing these methods are as described in Sections AK above.

M.M. Detection of a given base in the presence of a background guanine signal The presence of a given base in an oligonucleotide probe is detected even in the presence of a background signal generated from oxidation of guanine. This is because the detection of mismatch pairing depends on the ability to detect a given base in an oligonucleotide probe in the presence of four natural bases (A, T / U, C and G). Therefore, a given base must be capable of being oxidized more quickly than the other four bases.

  The present invention provides oligonucleotide probes useful for electrochemical detection of a given base in the presence of a background guanine signal. The oligonucleotide probe comprises any of the oligonucleotide probes set forth in Section B above, wherein at least one purine base in the oligonucleotide probe is of the formula I:

のプリン置換基である。オリゴヌクレオチドプローブは意図されたその結合の相手に従い、望みのように前記式の多数の塩基（例えば、１、２若しくは３から５，１０，若しくは１５まで又はそれ以上）を含みうる。かかるオリゴヌクレオチドプローブの特定例、及びその調製に有用なヌクレオチドは、式ＩＩ：

Purine substituents. Oligonucleotide probes may contain as many bases of the above formula as desired (eg, 1, 2, or 3 to 5, 10, or 15 or more), depending on the intended binding partner. Specific examples of such oligonucleotide probes, and nucleotides useful in their preparation, are those of formula II:

ここで：
Ｒ_１はＨＯ−Ｐ（Ｏ）（ＯＨ）−Ｏ−，ヌクレオチド、若しくはオリゴヌクレオチドであり；
Ｒ_２は−Ｈ，ヌクレオチド、若しくはオリゴヌクレオチドであり；
Ｒ_３は−Ｈ，−ＯＨ，ハロ（例えば、フロロ、クロロ）、アルコキシ（例えば、メトキシ又はエトキシ等のＣ１−Ｃ４アルコキシ）、アミノ、若しくはアジド；
Ｒ_４は−Ｏ−又は−ＣＨ_２−、である
の化合物である。

here:
R ₁ is HO—P (O) (OH) —O—, a nucleotide, or an oligonucleotide;
R ₂ is —H, a nucleotide, or an oligonucleotide;
R ₃ is —H, —OH, halo (eg, fluoro, chloro), alkoxy (eg, C1-C4 alkoxy such as methoxy or ethoxy), amino, or azide;
R ₄ is a compound of —O— or —CH ₂ —.

  Oligonucleotide probes as described in connection with Formula I and Formula II above are made according to known techniques and are improved in the disclosure of the examples set forth below as will be readily apparent to those skilled in the art. .

In one preferred embodiment of the compound of formula II, R ₁ is HO—P (O) (OH) —O—. In another preferred embodiment of the compound of formula I, R is H. When R ₁ is a nucleotide or oligonucleotide, the phosphodiester bond is up to the 3 ′ end. When R ₂ is a nucleotide or an oligonucleotide, the phosphodiester bond is up to 5 'end.

  Compounds of formula I are advantageously included as bases in the oligonucleotide probes utilized in the methods of the invention described in Sections AM above. The oligonucleotide probe will of course contain a large number of bases, but when the oligonucleotide probe is to be used for the detection of a given base in the presence of background guanine, it must contain at least one base of formula I There is. Oligonucleotide probes are base pairs close in length to 5, 10, 50 or 100. A specific example of a compound of formula II is 6-mercaptoguanosine 5'-monophosphate (6-S-GMP).

N. Electrode Structure An electrode useful for electrochemical detection of a predetermined base in a nucleic acid according to the above method comprises: (a) a conductive substrate having a working surface thereon; (b) a polymer layer connected to the working surface It consists of. The polymer layer binds to the nucleic acid (eg, hydrophobic interaction or any other suitable binding technique) and is permeable to the transition metal complex (ie, the transition metal complex is bound to the polymer To the nucleic acid). The conductive substrate is a metal substrate or a non-metal substrate including a semiconductor substrate (eg, gold, glassy carbon, indium doped tin oxide, etc.). The conductive substrate takes any physical form such as an elongated probe having an active surface formed at its end, or a flat sheet having an active surface formed on one side thereof. The polymer layer is connected to the working electrode by any suitable means, such as by clamping the polymer layer to the working surface, vapor deposition of a polymer solution on the electrode, or electrolytic polymerisation. Exemplary polymers include, but are not limited to, nylon, nitrocellulose, polystyrene, and poly (vinyl pyridine). The thickness of the polymer layer is not critical, but can be from 100 Å (A) to 1, 10 or 100 μm. The electrode can be used substantially in all the ways described in Sections AM. As a result, in general, the present invention provides a method of detecting a nucleic acid, the nucleic acid comprising at least one predetermined base, the method comprising: (a) an electrode formed on the working surface and the working surface A step of contacting a sample containing the nucleic acid with the electrode, and (b) oxidizing the nucleic acid with a predetermined base by an oxidation-reduction reaction. Reacting with a possible transition metal complex; (c) detecting a redox reaction by measuring current flow through the electrode; and (d) detecting the redox reaction with a predetermined base from the detected redox reaction. And determining the presence or absence of nucleic acids.

O. Microelectronic devices The advantage of the above techniques is that they are implemented using microelectronic devices. A microelectronic device useful for electrochemical detection of nucleic acid species in the above method comprises a microelectronic substrate having first and second opposite surfaces; a conductive electrode on the first surface; And an oligonucleotide capture probe immobilized on the first surface adjacent to the conductive electrode. The capture probe is placed close enough to the adjacent electrode so that the redox reaction occurring at the probe or at the nucleic acid of interest hybridized to the probe is detected at the adjacent electrode.

  In the embodiment shown in FIGS. 9 and 10, the microelectronic device 20 includes a number of separation electrodes 21 on a first opposite surface and a number of separation oligonucleotide capture probes immobilized adjacent to each separation electrode. Have Unlike others, the provision of multiple separate oligonucleotide probes, each with an associated electrode, provides a single, compact device that can detect changes in different hybridization results. Each electrode has a substantially suitable contact so that the device is wired or otherwise coupled with the electronic device necessary to effectively perform the detection and determination steps of the method described herein. 23 is substantially connected.

  The nucleic acid is fixed at an appropriate position on the substrate of the microelectronic device by a known technique. See, for example, US Pat. No. 5,405,783 to Pirrung et al. The substrate of the microelectronic device is a semiconductor (eg, silicon) or a non-semiconductor material (eg, glass) that can be processed using simple microelectronic technology. The electrode is a non-metallic conductive material such as metal or polycrystalline silicon. The electrodes can be formed using simple microelectronic processing techniques such as deposit etching. Suitable microelectronic structure changes and assembly techniques are well known to those skilled in the art. For example, S.M. M.M. Sze's VLSI Technology (1983); K. See Ghandhi's VLSI Fabrication Principles (1983).

The following examples are provided to illustrate the invention and should not be construed as limiting. In these examples, cm ² / s means square centimeters per second, M means molar concentration, M ⁻¹ s ⁻¹ means moles per second, eV means electron volts, V means volt, nm means nanometer, GMP means guanine 5'-monophosphate, ITO means an indium oxide electrode doped with tin. Cyclic voltammograms were obtained using a model 273A of EG + G Princeton Applied Research Potentiostat / Galvanostat according to known techniques. ITO working electrode is Delta Technology, Inc., 55960-1234, Minnesota, USA, 13960 North Street, Stillwater, 13960. Ltd .. Manufactured from ITO coated soda lime glass sheet with part number CH-501N-1514. The nylon membrane was manufactured by Amersham Corp., Clearbrook Drive 2636, Arlington Heights, 60005, Illinois, USA. As a high bond N + (HYBOND-N +) nylon membrane with catalog number RPN 1210B.

Measurement of cyclic voltammograms of Ru (bpy) ₃ ²⁺ Using contact enhancement generated by multiple turnovers of DNA oxidation due to the oxidized form of the metal complex observed during a single voltammetric sweep, A cyclic voltammogram of Ru (bpy) ₃ ²⁺ with and without bovine thymos DNA is shown in FIG. In FIG. 1, the uniform line represents a 50 μM Ru (bpy) ₃ ²⁺ scan at 25 mV / s in 700 mM NaCl / 50 mM sodium phosphate buffer, and the dotted line represents 50 μM Ru (bpy) ₃ ²⁺ and 1 represents a voltammogram of 3.0 mM (nucleotide) bovine thymos DNA. Since the diffusion coefficient of DNA (ie, 2.0 × 10 ⁻⁷ cm ² / s) is much smaller than that of the metal complex (8.0 × 10 ⁻⁶ cm ² / s), the voltammetry of the DNA-binding redox couple is Should be analyzed by a square diagram for combined and unbonded forms. This phenomenon generally results in a dramatically reduced current with respect to the binding form; however, at sufficiently high ionic strength ([Na ⁺ = 0.8 M]), the binding of the metal complex affects the current response. Is too weak. In this case, the current is analyzed by a simple EC ′ mechanism.

Ru (bpy) ₃ ²⁺ → Ru (bpy) ₃ ³⁺ (E)
Ru (bpy) ₃ ³⁺ + DNA → Ru (bpy) ₃ ²⁺ + DNAox (C ′)

Cyclic Voltammogram Analysis Cyclic voltammograms were analyzed using the DIGSIM ^™ data analysis package by fitting the complete current-potential curve with the subtracted background. The input parameters were E _{1/2 for the} metal complex and diffusion coefficient for the metal complex, all determined in the separation experiment. Therefore, the only parameter obtained from the fitting was the second-order rate constant k = 9.0 × 10 ³ M ⁻¹ s ⁻¹ for Equation 2. This same rate constant was determined over a wide range of sweep rates.

The rate constant for DNA oxidation by Ru (bpy) ₃ ³⁺ was confirmed in two separation experiments. First, square wave voltammograms were used to obtain quasi-first-order kobs for Equation 2 by fitting with the COOL ^TM algorithm. The COOLTM algorithm uses a fitting approach that is very different from DIGSIM ^™ , but the plot of k _obs against DNA is linear and is consistent with a rate constant obtained from fitting a cyclic voltammogram using DIGSIM ^™ . A rate constant k = 8.2 × 10 ³ M ⁻¹ s ⁻¹ was given. Second, a standard sample of Ru (bpy) ₃ ³⁺ was prepared and reacted directly with DNA in a stopped flow rapid scan. Overall analysis of the time-dependent spectra between 350 and 600 nm shows that Ru (bpy) ₃ ³⁺ is neatly converted to Ru (bpy) ₃ ²⁺ with a rate constant of 12 × 10 ³ M ⁻¹ s ⁻¹ without an intermediate. Showed that. Thus, the rate constant for DNA oxidation by Ru (bpy) ₃ ³⁺ is determined by two independent electrochemical measurements using dramatically different fitting protocols, and a non-electrochemical stopped flow technique using full visible spectral fitting. More clearly established.

Cyclic Voltammogram Analysis Given that the driving force for electron transfer is significantly less than the rearrangement energy (λ), the plot of RT ln k versus driving force (corrected for the work term coupled with reactant approach) Case), a straight line having a slope of 1/2 is formed. The rate constants for DNA oxidation by a number of Metal (bpy) ₃ ³⁺ derivatives with different redox potentials are shown in Table 1 below.

Marcus theory describes the driving force dependence of the electron movement speed, and the absolute speed constant is:
k = νexp [−β (r−r _o )] exp [− (ΔG + λ) ² / 4λRT]
Where ν is the rate constant in diffusion rate (10 ¹¹ M ⁻¹ s ⁻¹ ), r is the distance between the reactant and product in the activated complex, and r ₀ is the closest distance between the reactant and product, and β indicates the influence of the intervening medium. Incorporation of a guanine donor into the interior of the double helix imposes a finite distance beyond which electrons must tunnel to the bound metal complex, ie r ≠ r ₀ . However, if guanine 5′-monophosphate (GMP) is used as the electron acceptor, direct diffusion of guanine with a metal complex is possible (r = r ₀ ). For Fe (bpy) ₃ ³⁺ and GMP, the rate constant measured by stopped flow is 2.6 × 10 ³ M ⁻¹ s ⁻¹ . Known values of λ for the relevant reactions are in the range of 1-1.5 eV, giving a ΔG for guanine ^{+ / 0} linkage of 1.1 ± 0.1 V.
Table 1. Rate constants for oxidation of guanine in DNA oligomers by Ru (bpy) ₃ ²⁺

^ａ 速度定数を決めるために使用されるＤＮＡ濃度はグアニンヌクレオチドのモルに基づく。
^ｂ 溶媒を通るトンネリングの確立された距離。β（Ｈ_２Ｏ）＝３Å^１及びｋ_ＳＳ＝１．８×１０^５Ｍ^−１Ｓ^−１で、ｋ／ｋ_ＳＳ＝ｅｘｐ［−βΔｒ］に従い計算された距離。
^ｃ 速度定数はグアニン濃度に相関するので、ＧＧ不正対合に対して観測された速度は一つのグアニンを含む他のオリゴマーに関して規格化された。

DNA concentrations used to determine ^a rate constant based on the moles of guanine nucleotides.
^b Established distance of tunneling through the solvent. β _(H 2 O) = at 3 Å ¹ and _{^{k SS = 1.8 × 10 5 M}} -1 S -1, is calculated according to k / k SS = exp [-βΔr ] distance.
^{Since the c} rate constant correlates with guanine concentration, the rate observed for GG mismatch pairings was normalized with respect to other oligomers containing one guanine.

In FIG. 2 there is a cyclic voltammogram of Ru (bpy) ₃ ^{2+ in} the presence of single strand 5′-AAATATAGTATAAAAA (C) and its hybrid to its complementary strand. In FIG. 2, the sweep rate is 25 mV / s and (A) is 25 μM Ru (bpy) ₃ ²⁺ +100 μM double-stranded fully hybridized DNA (in guanine nucleotides) (5′-AAATATAGTATAAAAA) · (3 ′ -TTTATACATCATTTTT), (B) represents GA illegal pairing (5'-AAATATAGTATAAAAA) (3'-TTTATAATAATTTT), and (C) is a single strand containing one guanine nucleotide (5'-AAATATAGTAATAAA). Ru (bpy) ₃ ²⁺ is represented. For GMP, the rate constant of r = r ₀ and 1.8 × 10 ⁵ M ⁻¹ s ⁻¹ for a single strand gives ΔG (guanine ^{+ / 0} ) = 1.1 V and λ = 1.3 eV, Consistent with values from GMP oxidation. While there was a dramatic increase for the single strand, it resulted in a 4-fold decrease in current for hybrid formation, and only a slight increase was observed at this sweep rate for the fully hybridized duplex. Metal complexes such as Ru (bpy) ₃ ²⁺ are known to bind to DNA in a small groove, and thus a 150-fold slower rate constant (1.2 × 10 ³ M ⁻¹ s ⁻ for duplex oxidation). ¹ ) is obtained from the distance between the guanine residue and the surface bound complex. When docked in a small groove in a metal complex, guanine and the metal complex do not come into intimate contact, and the electrons need to be tunneled through a solvent that separates the guanine residue from the metal complex. Tunneling through water is much less efficient than through non-polar media, and the value of β for water is established to be about 3Å- ¹ . So the tunneling distance is:
k / kss = exp (−βΔr)
Where Δr is the change in distance in the duplex compared to the single strand. From this analysis, Δr for a fully hybridized duplex is 1.7 Å.

Large values for water indicate that large differences in electron transfer rate constants are caused by very small changes in the tunneling distance, which in turn can reflect small perturbations in the DNA structure. Also shown in FIG. 2 is a voltammogram of Ru (bpy) ₃ ²⁺ in the presence of the same duplex in which the GC base pair is replaced by GA mispairing. Incorporation of GA mismatch pairing results in a two-fold increase in raw current compared to the standard duplex, which is interpreted as a 16-fold change in the rate constant (k _GA = 1.9 × 10 ⁴ M ⁻¹ s ⁻¹ ). The velocity data for all single stranded, fully hybridized duplex, and 3 GX mismatches are displayed in Table 1. Also shown is the calculated tunneling distance Δr compared to the single strand. As expected, the guanine residue in the G-purine mismatch is more to the metal complex than in the GT mismatch where the two bases are linked by two hydrogen bonds in a still unstable pair. Easy to approach. However, GT mispairing still causes a 4-fold change in the rate constant that can be easily detected. Therefore, the oxidation rate constant follows the trend G (single chain)>GA>GG>GT> GC. The ability to distinguish each of these mismatches from one another is due to hybridization mismatch sensitive detection that is sensitive to even single base pair mismatches at base pairs adjacent to a given base. Provides the basis for

Improved base to avoid oxidation in the probe strand: substitution of inosine for guanine The cyclic voltammogram uses indium tin oxide (ITO) working electrode (area = 0.32 cm ² ), Pt-wire counter electrode, and Ag / AgCl reference electrode And was acquired. In FIG. 5, a sample containing 25 μM Ru (bpy) ₃ ²⁺ and 75 μM oligonucleotide dissolved in 50 mM Na-phosphate buffer (pH 7) containing 0.70 M NaCl was scanned at 25 mV / s. . In FIG. 5, (A) shows no added oligonucleotide, (B) contains 75 [mu] M d [5'-TTTTATATACTATTTT], and (C) shows a 75 [mu] M oligomer from B and d [5'-GGGAAATATAGTATAAAAGGG]. Accompanied by a hybrid. The working electrode is indium oxide doped with tin, the reference electrode is Ag / AgCl, and the counter electrode is a Pt wire. The secondary structure of the hybrid from C is shown in the figure. In FIG. 6, 50 μM Ru (bpy) ₃ ²⁺ and 0.3 mM dissolved in a buffered aqueous solution containing 700 mM NaCl and 50 mM Na-phosphate buffer (pH = 6.8, [Na ⁺ = 780 mM]). Samples containing either 5′-GMP or 5′-IMP were scanned from 0.0 V to 1.4 V at 2.5 mV / s. Scanning mononucleotides in the absence of Ru (bpy) ₃ ²⁺ showed no clear oxidation current. A freshly cleaned ITO electrode was used for each experiment, and a buffer-only background scan was subtracted from subsequent scans. Second order guanine oxidation rate constants were determined by fitting cyclic voltammetric data to a two-step mechanism using the DIGSIM ™ software package. All parameters except the oxidation rate were determined from the voltammogram of the metal complex alone at the same electrode. 5′-GMP was purchased from Sigma and 5′-IMP was purchased from U.S. S. Both were purchased from Biochemical and both were used without further purification. Oligonucleotides were prepared in UNC Department of Pathology and passed through a 3000-molecular weight cut-off filter to remove mononucleotides. Purity was assessed by reverse phase HPLC. Concentrations are reported in Fasman, G .; D. CRC Handbook of Biochemistry and Molecular Biology; Vol. 1, 1975, determined from light absorption at 260 nm as described in CRC Press, Boca Raton, Florida. The hybrid of FIG. 5 was prepared by heating the complementary strand at 90 ° C. for 5 minutes and slowly cooling to 250 ° C. over 2 hours.

  These data indicate that inosine can be substituted for guanine in the probe to provide a redox-inactive probe strand.

Improved base to avoid oxidation in the probe strand: 7-deaza-guanine This example is that 7-deaza-guanine is used as an improved base to replace guanine to provide a redox-inactive probe strand. This is carried out in substantially the same manner as in Example 4 above.

7-deaza-guanine is oxidized at a rate of only 10 ³ M ⁻¹ s ⁻¹ , which is slower in magnitude to a square order than guanine and is slow enough to provide a redox-inactive probe strand.

Detection using bovine thymos DNA bound to a nylon membrane attached to an ITO electrode. The nylon film fits in an electrochemical cell and forms a circular shape with a diameter of approximately 6 mm to cover a portion of the ITO electrode exposed to the solution. disconnected.

For experiments that obtain only cyclic voltammograms of metal complexes, the ITO electrode is initially conditioned with buffer. The nylon disc (without DNA) is then inserted into the electrochemical cell and 200 μL of a 200 μM metal complex solution is pipetted into the cell. For the Os (bpy) ₃ ²⁺ experiment, a 6 minute equilibration time was used prior to electrochemical analysis. For Ru (bpy) ₃ ²⁺ experiments, a 15 minute equilibration time was used prior to electrochemical analysis. Cyclic voltammograms were acquired using a PAR 273A potentiostat with a sweep rate of 25 mM / s.

For DNA experiments, a DNA-absorbing nylon disk is inserted into the electrochemical cell after adjusting the ITO electrode in a suitable buffer. 200 μL of a 200 μM metal complex solution in an appropriate buffer is pipetted into the cell and after an appropriate equilibration time (6 minutes for Os (bpy) ₃ ²⁺ and 15 minutes for Ru (bpy) ₃ ²⁺ ) Cyclic voltammograms are taken at a sweep rate of 25 mM / s. The nylon disk is immersed in a solution of 5.8 mM bovine thymos DNA in water for approximately 5 minutes. The change in immersion time was investigated in the range of 5 minutes to 18 hours. DNA binds quickly (within a few minutes) with nylon film, so a short soak time is typically used. Under low salt conditions, 50 mM Na-phosphate buffer (pH = 6.8, [Na ⁺ ] = 80 mM) is used. Under high salt conditions, a solution of 50 mM Na-phosphate buffer and 700 mM NaCl (pH = 6.8, [Na ⁺ ] = 780 mM) is used.

A cyclic voltammogram of Ru (bpy) ₃ ²⁺ at the nylon-ITO electrode is shown in FIG. The dotted line shows the voltammogram when the nylon membrane is immersed in bovine thymos DNA before being attached to the electrode. There is a large contact current for the DNA labeled membrane corresponding to that observed in solution. The experiment shows that Ru (bpy) ₃ ²⁺ diffuses freely in nylon film and that DNA diffusion is not necessary to achieve contact current. FIG. 11 also shows that larger contact currents are observed at lower salt concentrations due to increased interaction between the mediator and immobilized DNA.

FIG. 12A shows the same experiment using Os (bpy) ₃ ²⁺ as a mediator. Osmium complexes do not oxidize guanine, so the current increase observed in the presence of guanine must result from preconcentration of mediators by DNA bond formation. In fact, the current for Os (bpy) ₃ ²⁺ is lower in the presence of DNA at the nylon electrode than in the absence of DNA. The experiment shows that the increased current for Ru (bpy) ₃ ²⁺ when DNA is bound to the nylon electrode is solely due to the proprietary catalytic reaction and not due to differences in trivalent bond formation. The effect of salt concentration is shown in FIG. 12B and is small compared to the effect of large salts observed for the catalytic reaction.

  Binding of the DNA to the nylon membrane attached to the ITO electrode showed that the DNA was detected even in examples where the DNA was not diffused but the mediator was. This discovery allows the detection of DNA when the immobilized probe is close enough to the electrode so that the probe-object hybrid is in the diffusion layer of the mediator.

Detection of RNA bound to nylon membrane attached to ITO electrode The experiment is described in Example 6 except that tRNA from Bakers Yeast (obtained from Sigma) is used instead of bovine thymos DNA. To be implemented. Nylon film discs were immersed in a solution of tRNA as described in Example 6.

Cyclic voltammetry in the presence of Ru (bpy) ₃ ²⁺ is shown in FIG. As with DNA, contact current is observed with both buffers and is greater with low concentrations of salt. Since tRNA does not bind to cations like DNA and therefore the salt effect is not very dramatic, the observed difference in current between high and low salt concentrations is as dramatic as that observed with the DNA of Example 6. is not.

  The results in FIG. 13 show that RNA can be detected in the same way as for DNA, which occurs because both RNA and DNA contain guanine. The chemical composition of the sugar-phosphate backbone does not affect the contact current. Based on this observation, single and double stranded DNA and RNA, as well as single or double stranded, including other improved backbones of PNA, such as carbocycles, phosphorothioates, or other substituted ribose linkages. DNA-RNA hybrids can be detected.

Detection of RNA For quantitative detection of RNA, a DNA (or RNA, PNA, or other alternative backbone) probe is immobilized on a solid support. The probe is modified to be redox inactive by substitution of inosine or 7-deaza-guanine instead of guanine in the probe strand. The immobilized probe is then contacted with a solution of the RNA of interest (eg, from HIV or Hepatitis C). The solid surface then contains a redox inert, immobilized probe that is hybridized with a strand of RNA. The solid surface is then contacted with a solution of Ru (bpy) ₃ ²⁺ and the cyclic voltammogram of the mediator is measured. The contact current indicates the hybridization result, and the magnitude of the current is used to quantify the RNA strands bound based on the number of known guanines in the strand.

For RNA mispair detection, a DNA (or RNA, PNA, or other alternative) probe is immobilized on a solid support. Certain bases in the probe strand are more easily oxidized than other bases. The surface is contacted with a solution of RNA of interest followed by a solution of Ru (bpy) ₃ ²⁺ or other mediator. The result of hybridization (complete pairing, non-pairing or mispairing) is then determined at a given base in the same way as for DNA.

The method for detecting a predetermined sequence of bases is performed as described in Example 3. The four cyclic voltammograms placed in FIG. 14 show that the current due to 5′-G is much smaller than that for 5′-GG which is much smaller than for 5′-GGG. A very large increase in this current is observed for both single and double strands containing GG and GGG sequences. As shown in FIG. 15, the increase in current due to the addition of G to the same chain is much smaller if G is placed in dots, so the increase in current is not simply due to an increase in the number of G. Since 5′-G of GGG is oxidized much more easily than a single G, it is possible to select a mediator capable of oxidizing GGG rather than G (having a lower redox potential).

A cyclic voltammogram of Ru (4,4′-dimethyl-bipyridine) ₃ ²⁺ is shown in FIG. 16 with repeated scans in the presence of a single G oligonucleotide and a GGG oligonucleotide. As shown, contact current is observed only in the presence of GGG oligonucleotide. This example demonstrates the ability to modulate the potential of the mediator such that a more easily oxidized sequence can be detected in the presence of background guanine. The same scheme can be applied to detect a single synthetic base derived to be more easily oxidized than guanine.

  The examples show that the potential of the mediator can be lowered and the bases or base sequences that can be more easily oxidized can be identified.

Background Detection of certain guanine derivatives in the presence of natural guanine 6-mercaptoguanosine 5'-monophosphate (6-S-GMP) disodium salt

が購入により入手可能な６−メルカプトグアノシン（Ｓｉｇｍａから）のリン酸化により調製される。リン酸化は、Ｍ． Ｙｏｓｈｉｋａｗａ他のＢｕｌｌ． Ｃｈｅｍ． Ｓｏｃ． Ｊｐｎ ４２巻の３５０５頁（１９６９年）の製法に従いＰＯＣｌ_３を使用して行なわれる。６−Ｓ−ＧＭＰの二ナトリウム塩はボルタンメトリー分析の前にＨＰＬＣにより精製される。サイクリックボルタモグラムは、イノシン−５’−モノホスフェートの例のように高いイオン強度で実施される。作用電極は６−Ｓ−ＧＭＰの直接酸化を防ぐために表面に取り付けられたハイボンドＮ＋ナイロン膜を有するＩＴＯである。対極はＰｔワイヤである。参照電極はＡｇ／ＡｇＣｌである。掃引速度は２５ｍＶ／ｓである。

Is prepared by phosphorylation of commercially available 6-mercaptoguanosine (from Sigma). Phosphorylation is carried out by M.M. Yoshikawa et al. Bull. Chem. Soc. Jpn 42, 3505 (1969), using POCl ₃ . The disodium salt of 6-S-GMP is purified by HPLC prior to voltammetric analysis. Cyclic voltammograms are performed at high ionic strength as in the example of inosine-5′-monophosphate. The working electrode is ITO with a high bond N + nylon film attached to the surface to prevent direct oxidation of 6-S-GMP. The counter electrode is a Pt wire. The reference electrode is Ag / AgCl. The sweep speed is 25 mV / s.

Four cyclic voltammogram results are shown in FIG. 17, and curve A represents Ru (4,4′-Me ₂ -bpy) ₃ ²⁺ alone (4,4′-Me ₂ -bpy = 4,4′-dimethyl). -Bipyridine). With the addition of 5′-GMP, no increase in Ru (Me ₂ bpy) ₃ ²⁺ waves is observed; however, the addition of 6-mercaptoguanosine 5′-monophosphate (6-S-GMP) results in a dramatic current increase. Guide (curve B). The peak current in the presence of 5′-GMP is identical to that of curve A. The data show that 6-mercaptoguanosine base can be detected in the presence of background natural guanine.

Detection of DNA hybridization with a predetermined base on the target strand A nylon membrane (High Bond N +, Amersham, 480-600 μg / cm ² ) is cut into a circular shape with a diameter of approximately 6 mm. The nylon disc is placed in a concentrated solution of polycytidic acid (available from Sigma) in water and soaked for 1 hour. The disc is then removed from the polycideic acid (poly [C]) solution, placed on parafilm and dried. As the disc dries, an additional 15 μl of poly [C] solution is added to the film in three 5 μl aliquots. The disc is completely dried. The dried nylon disc is then washed with a low salt buffer (50 mM Na-phosphate, pH = 6.8, [Na ⁺ ] = to remove poly [C] that is not strongly bound during the dipping process. In 80 mM).

  As a control experiment, a poly [C] -impregnated disk is passed through a pseudohybridization process that is not exposed to added nucleic acid but undergoes all other hybridization steps. The disc is placed in 400 μl of MilliQ water and heated at 48 ° C. for 1 hour and cooled to room temperature. The disc is removed from the water and washed with a low salt buffer prior to electrochemical analysis. The disc thus prepared represents the background scan (A) of both FIGS.

  The poly [C] soaked disc is placed in an aqueous solution of 400 μl polyguanic acid (available from Sigma), heated at 48 ° C. for 1 hour, and cooled to room temperature. The disc is then removed from its polyguanic acid (poly [G]) solution and washed with a low salt buffer prior to electrochemical analysis. Bovine thymos DNA in water (available from Sigma) is denatured (melted) by heating to 90 ° C. for 10 minutes. A disc impregnated with poly [C] is placed in a denatured bovine thymos DNA solution, heated at 48 ° C. for 1 hour, and cooled to room temperature. The disc is then removed from the bovine thymos DNA solution and washed with a low concentration of salt buffer prior to electrochemical analysis. As a control, a non-soaked poly [C] disc also received the same process. Binding and detection of bovine thymos DNA solution by absorption into the nylon film (not by hybridization) is observed in the control membrane.

After conditioning the ITO electrode with a low concentration salt buffer, the nylon disk treated as described above is inserted into the electrochemical cell. 200 μl of 200 μM Ru (bpy) ₃ ²⁺ solution is pipetted into the cell and a cyclic voltammogram is taken after a 15 minute equilibration time. The sweep speed is 25 mV / sec.

A cyclic voltammogram is reported in FIG. The probe sequence poly [C] is immobilized on a high bond N + nylon membrane and a hybrid formation protocol is performed in buffer (“pseudohybridization”). The membrane is attached to the ITO working electrode, and a cyclic voltammogram of Ru (bpy) ₃ ²⁺ is obtained (A). The membrane is immersed in a solution of poly [G] and hybridization is performed according to the same protocol. The cyclic voltammogram of Ru (bpy) ₃ ²⁺ is then measured (B) and a large current increase is obtained for the catalytic oxidation of the hybrid poly [G] object. As shown in FIG. 19, the analysis is specific for a well-defined sequence. FIG. 19 compares voltammetry for poly [C] membranes when the hybridization process is performed in buffer (A) or in a solution of single-stranded bovine thymos DNA (B). FIG. 19 shows that no current increase can be obtained if the target array is not present.

FIG. 20 shows cyclic voltammograms of glassy carbon electrodes with the nylon film attached before (A) and after (B) DNA immobilization on the nylon film. Or “CV”).

A nylon membrane (Zeta-probe, Bio-Rad, 80-100 μg / cm ² ) was cut into a circle with a diameter of approximately 5 mm. The formed nylon disk covers the glassy carbon electrode surface and is held in position by a resin sleeve. For experiments where only CV of the metal complex was obtained, the glassy carbon electrode was first conditioned with a low salt concentration, 50 mM Na-phosphate buffer (pH = 6.8, [Na ⁺ ] = 80 mM). It was done. A nylon disk (no DNA) was then attached to the electrode and 400 μl of 200 μM Ru (bpy) ₃ ²⁺ solution was pipetted into the electrochemical cell. A 15 minute equilibration time was used prior to electrochemical analysis. Cyclic voltammograms were acquired using a PAR 273A potentiostat with a sweep rate of 25 mV / sec. In a typical DNA experiment, a glassy carbon electrode is first conditioned in low salt Na-phosphate buffer. The nylon disc was immersed in a solution of 5.8 mM bovine thymos DNA dissolved in water for approximately 5 minutes. The disc was then removed from the solution and placed on the glassy carbon electrode using a sleeve to hold it in place. 400 μl of 200 μM (Ru (bpy) ₃ ²⁺ solution was pipetted into the electrochemical cell and a cyclic voltammogram was taken with a sweep rate of 25 mV / sec after a 15 minute equilibration time.

  The above description is illustrative of the present invention and should not be construed as limiting. The invention is defined by the following claims, along with the equivalents of the claims to be included therein.

FIG. 6 is a graph showing cyclic voltammograms of Ru (bpy) ₃ ²⁺ with and without bovine thymos DNA. FIG. 5 is a graph showing a cyclic voltammogram of Ru (bpy) ₃ ²⁺ in the presence of 5′-AAATATAGTATAAAAA as hybridized to single strand (C) and complementary strand (A & B). FIG. 2 is a schematic diagram of one illustrative apparatus useful for performing the method of the present invention. It is a schematic diagram of a detection method particularly useful for quantitative detection of DNA in which a predetermined base is placed on a target nucleic acid. FIG. 6 is a graph showing a cyclic voltammogram of Ru (bpy) ₃ ²⁺ (25 μM) at a sweep rate of 25 mV / s in a 50 mM sodium phosphate buffer containing 0.7 M NaCl at pH 7. FIG. Structure of inosine and guanine are shown in the ^{_{figure, (A) Ru (bpy)}} 3 2+ (25μM), Ru with (B) an inosine 5'-monophosphate ^{_{(0.3mM) (bpy) 3 2+}} (25μM ), And (C) a cyclic voltammogram of Ru (bpy) ₃ ²⁺ (25 μM) with guanosine 5′-monophosphate (0.3 mM). FIG. 5 is a schematic diagram of an alternative embodiment of the invention of FIG. 4 where a given base is on the extension product of the terminal transferase. FIG. 5 is a schematic diagram of an alternative embodiment of the invention of FIG. 4 implemented in a sandwich analysis format. 1 is a schematic top view of a micropotential device useful for carrying out the method of the present invention. FIG. FIG. 10 is a side view of a portion of the apparatus shown in FIG. 9. _Ru in buffer dipped nylon ^{(bpy) 3 2+ (200μM)} , Ru (bpy) 3 2+ in the DNA absorption nylon high salt (700 mM added NaCl) of buffer (200 [mu] M), and low salt FIG. 5 is a graph showing a cyclic voltammogram of Ru (bpy) ₃ ²⁺ (200 μM) with DNA-absorbing nylon in buffer (ie, no NaCl added) using a nylon modified ITO electrode. FIG. 6 is a graph showing a cyclic voltammogram with 700 mM added NaCl, which is a cyclic voltammogram of Os (bpy) ₃ ²⁺ (200 μM) using a nylon modified ITO electrode immersed in buffer or DNA. FIG. 2 is a graph showing a cyclic voltammogram with no NaCl added, which is a cyclic voltammogram of Os (bpy) ₃ ²⁺ (200 μM) using a nylon modified ITO electrode immersed in buffer or DNA. _Ru in buffer dipped nylon ^{(bpy) 3 2+ (200μM)} , Ru (bpy) 3 2+ in the tRNA absorption nylon high salt (700 mM added NaCl) of buffer (200 [mu] M), and low salt FIG. 6 is a graph showing a cyclic voltammogram on a nylon modified ITO electrode showing a cyclic voltammogram of Ru (bpy) ₃ ²⁺ (200 μM) with tRNA-absorbing nylon in buffer (ie, no NaCl added). Ru (bpy) ₃ ²⁺ (25 μM) alone with a sweep rate of 25 mV / s and 5′-AAATATAGn TATAAAA (100 μM in the chain) with n = 1 (G), 2 (GG), or 3 (GGG) It is a graph which shows the cyclic voltammogram of what accompanies. Cyclic voltammograms of Ru (bpy) ₃ ²⁺ (25 μM) alone with a sweep rate of 25 mV / s and with 5′-AAATAT (AGT) nATAAAA (100 μM in chain) with n = 1, 2, or 3. It is a graph to show. 25 μM ruthenium (4,4′-dimethylbipyridine) ₃ ²⁺ (or “Ru (4,4′-Me ₂ -bpy) ₃ ²⁺ ”) alone (uniform) and 5 ′ with a sweep rate of 25 mV / s FIG. 5 is a graph showing cyclic voltammograms of those with -AAATATAGTATAAAAA (dots) and 5′-AAATATAGGGTAATAAA (dashes) (100 μM in chain). Curve (A) represents a single Ru (4,4′-Me ₂ -bpy) ₃ ²⁺ and curve (B) represents Ru (4 in the presence of 0.70 mM 6-mercaptoguanosine 5′-monophosphate. , 4′-Me ₂ -bpy) ₃ ²⁺ with 0.2 mM Ru (4,4 in 50 mM sodium phosphate buffer (pH 7) with 0.7 M NaCl at a sweep rate of 25 mV / s. 4′-Me ₂ -bpy) ₃ ²⁺ is a graph showing a cyclic voltammogram. The membrane is impregnated with poly [C] and subjected to a hybridization protocol in buffer (A) and in a concentrated solution of poly [G] (B) at 200 μM at the ITO working electrode fitted with a high bond N + nylon membrane. is a graph showing cyclic voltammograms of ru ^{_(bpy) 3 2+.} The membrane is impregnated with poly [C] and subjected to a hybridization protocol in buffer [A] and in a concentrated solution of denatured bovine thymos DNA, 200 μM Ru (bpy) at the ITO working electrode fitted with a high bond N + nylon membrane. ₃ is a graph showing a ₃ ²⁺ cyclic voltammogram. Nylon without DNA modified glassy electrode (A) or cyclic voltammogram of 200 μM Ru (bpy) ₃ ²⁺ after absorption of DNA into the nylon film (B) (sweep rate = 25 mV / s) It is a graph which shows.

Claims (16)

(A) contacting the DNA sample with an oligonucleotide probe to form hybridized DNA;
(B) The oligonucleotide probe has at least one predetermined base, and the hybridized DNA reacts with a transition metal complex capable of oxidizing the predetermined base in the oligonucleotide probe by an oxidation-reduction reaction. And wherein there is an electron transfer from the predetermined base to the transition metal complex, and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle ;
(C) detecting the redox reaction;
(D) determining the presence or absence of hybridizing DNA from the detected redox reaction at the predetermined base;
(E) A method for detecting DNA hybridization comprising the step of identifying a base paired with the predetermined base or a base paired with a base adjacent to the predetermined base. (A) contacting the DNA sample with an oligonucleotide probe to form hybridized DNA;
(B) The oligonucleotide probe has at least one predetermined base, and the hybridized DNA reacts with a transition metal complex capable of oxidizing the predetermined base in the oligonucleotide probe by an oxidation-reduction reaction. And wherein there is an electron transfer from the predetermined base to the transition metal complex, and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle ;
(C) detecting the redox reaction;
(D) measuring the reaction rate of the detected redox reaction;
(E) comparing the measured reaction rate with the rate of redox reaction of the transition metal complex with single-stranded DNA;
(F) determining whether the measured reaction rate is the same as the rate of the redox reaction of the transition metal complex with the single-stranded DNA;
(G) A method for detecting DNA hybridization comprising the step of identifying a base paired with the predetermined base or a base paired with a base adjacent to the predetermined base. (A) a plurality of DNA sample containers;
(B) a sample handling means for carrying the plurality of DNA sample containers;
(C) oligonucleotide probe delivery means for delivering the oligonucleotide probe to each of the DNA sample containers;
(D) transition metal complex delivery means for delivering the transition metal complex to each of the plurality of DNA sample containers;
(E) a redox reaction detector for detecting a redox reaction;
(F) a device for detecting DNA hybridization , comprising means for measuring the redox reaction rate of the detected redox reaction , wherein electrons are transferred from a predetermined base of the oligonucleotide probe to the transition metal complex. An apparatus wherein there is transfer and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle. (A) a DNA sample container;
(B) oligonucleotide probe delivery means for delivering a plurality of oligonucleotide probes to the DNA sample container;
(C) a transition metal complex delivery means for delivering the transition metal complex to the DNA sample container;
(D) a redox reaction detector for detecting a redox reaction;
(E) a device for detecting DNA hybridization , comprising means for measuring the redox reaction rate of the detected redox reaction , wherein electrons are transferred from a predetermined base of the oligonucleotide probe to the transition metal complex. An apparatus wherein there is transfer and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle. (A) contacting the DNA sample with the oligonucleotide probe to form a hybridized DNA, wherein the oligonucleotide probe comprises a predetermined base having a unique oxidation potential;
(B) The oligonucleotide probe has a predetermined number of predetermined bases, and the hybridized DNA is reacted with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe by an oxidation-reduction reaction. And there is a transfer of electrons from the predetermined base to the transition metal complex, and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle ;
(C) detecting a redox reaction;
(D) measuring the reaction rate of the detected redox reaction;
(E) A method for sequencing DNA comprising the step of identifying a base paired with the predetermined base. (A) contacting the RNA sample with an oligonucleotide probe to form hybridized RNA;
(B) The oligonucleotide probe has at least one predetermined base, and the hybridized RNA reacts with a transition metal complex capable of oxidizing the predetermined base in the oligonucleotide probe by an oxidation-reduction reaction. And wherein there is an electron transfer from the predetermined base to the transition metal complex, and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle ;
(C) detecting a redox reaction;
(D) determining the presence or absence of hybridizing RNA from the detected redox reaction at the predetermined base;
(E) A method for detecting RNA hybridization comprising the step of identifying a base paired with the predetermined base or a base paired with a base adjacent to the predetermined base. (A) contacting the RNA sample with an oligonucleotide probe to form hybridized RNA;
(B) The oligonucleotide probe has at least one predetermined base, and the hybridized RNA reacts with a transition metal complex capable of oxidizing the predetermined base in the oligonucleotide probe by an oxidation-reduction reaction. And wherein there is an electron transfer from the predetermined base to the transition metal complex, and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle ;
(C) detecting the redox reaction;
(D) measuring the reaction rate of the detected redox reaction;
(E) comparing the measured reaction rate with the rate of redox reaction of the transition metal complex with single-stranded RNA;
(F) determining whether the measured reaction rate is the same as the rate of the redox reaction of the transition metal complex with the single-stranded RNA;
(G) A method for detecting RNA hybridization comprising the step of identifying a base paired with the predetermined base or a base paired with a base adjacent to the predetermined base. (A) contacting the RNA sample with the oligonucleotide probe to form a hybridized RNA, wherein the oligonucleotide probe comprises a predetermined base having a unique oxidation potential;
(B) The oligonucleotide probe has a predetermined number of predetermined bases, and the hybridizing RNA is reacted with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe by an oxidation-reduction reaction. And there is a transfer of electrons from the predetermined base to the transition metal complex, and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle ;
(C) detecting a redox reaction;
(D) measuring the reaction rate of the detected redox reaction;
(E) A method for sequencing RNA comprising the step of identifying a base paired with the predetermined base. The nucleic acid comprises at least one predetermined base;
(A) reacting the nucleic acid with a complementary nucleic acid to form a hybridizing nucleic acid;
(B) reacting the nucleic acid with a transition metal complex capable of oxidizing the predetermined base by an oxidation-reduction reaction, wherein there is transfer of electrons from the predetermined base to the transition metal complex, and a catalytic cycle A reduced form of the transition metal complex is regenerated as part of
(C) detecting the redox reaction;
(D) determining the presence or absence of the nucleic acid from the detected redox reaction at the predetermined base;
(E) A method for detecting a nucleic acid, comprising: identifying a base paired with the predetermined base or a base paired with a base adjacent to the predetermined base. The target nucleic acid contains at least one predetermined base,
(A) The target nucleic acid contains at least 10 or more of the predetermined base as compared to the oligonucleotide probe; the test sample is contacted with the oligonucleotide probe that specifically binds to the target nucleic acid to form a hybridized nucleic acid Stages;
(B) contacting the hybridizing nucleic acid with a transition metal complex that oxidizes the predetermined base by an oxidation-reduction reaction, wherein there is electron transfer from the predetermined base to the transition metal complex, In part, the reduced form of the transition metal complex is regenerated ;
(C) detecting the presence or absence of the redox reaction bound to the hybridizing nucleic acid;
(D) determining the presence or absence of the nucleic acid of interest in the test sample from the detected redox reaction at the predetermined base, in the test sample presumed to contain the same A method for detecting the presence or absence of the target nucleic acid. The target nucleic acid contains at least one predetermined base,
(A) contacting the test sample with the oligonucleotide probe that does not have the predetermined base and specifically binds to the nucleic acid of interest to form a hybridizing nucleic acid;
(B) contacting the hybridizing nucleic acid with a transition metal complex that oxidizes the predetermined base by an oxidation-reduction reaction, wherein there is electron transfer from the predetermined base to the transition metal complex, In part, the reduced form of the transition metal complex is regenerated ;
(C) detecting the presence or absence of a nucleic acid reduction reaction associated with the hybridizing nucleic acid;
(D) determining the presence or absence of the nucleic acid of interest in the test sample from the detected redox reaction at the predetermined base, in the test sample presumed to contain the same A method for detecting the presence or absence of the target nucleic acid. The target nucleic acid contains at least one predetermined base,
(A) contacting the test sample with the oligonucleotide probe that does not have the predetermined base and specifically binds to the nucleic acid of interest to form a hybridizing nucleic acid;
(B) contacting the hybridizing nucleic acid with a transition metal complex capable of oxidizing the predetermined base by an oxidation-reduction reaction, wherein there is an electron transfer from the predetermined base to the transition metal complex; Regenerating the reduced form of the transition metal complex as part of a catalytic cycle ;
(C) detecting the presence or absence of the redox reaction bound to the hybridizing nucleic acid;
(D) determining the presence or absence of the nucleic acid in the test sample from the redox reaction detected at the predetermined base, wherein the predetermined base in the target nucleic acid is guanine; The nucleic acid of interest comprises cytosine and the oligonucleotide probe comprises a surrogate base that binds to cytosine in the hybridizing nucleic acid; the surrogate base is selected from the group consisting of inosine and 7-deaza-guanine; A method for detecting the presence or absence of a target nucleic acid in a test sample presumed to contain. (A) contacting the test sample with the oligonucleotide probe that has an end that is blocked for extension by terminal transferase and specifically binds to the nucleic acid of interest to form a hybridizing nucleic acid; ;
(B) contacting the hybridizing nucleic acid with a solution containing a predetermined base in the presence of a terminal transferase in order to produce the extension product of the target nucleic acid, which is an extension product consisting of a predetermined base;
(C) contacting the oligonucleotide probe with a transition metal complex that oxidizes a predetermined base by an oxidation-reduction reaction , wherein electrons are transferred from the predetermined base to the transition metal complex, A reduced form of the transition metal complex is regenerated as part ;
(D) detecting the presence or absence of the redox reaction;
(E) an object in the test sample presumed to contain the same, comprising the step of determining the presence or absence of the target nucleic acid in the test sample from the detected redox reaction at the predetermined base A method for detecting the presence or absence of a nucleic acid. (A) the oligonucleotide capture probe specifically binds to the target nucleic acid, and provides the oligonucleotide capture probe;
(B) contacting the test sample with the capture probe to form a hybridizing nucleic acid;
(C) the oligonucleotide signal probe specifically binds to the nucleic acid of interest, and the signal probe contains at least one predetermined base to produce a hybridized nucleic acid sandwich; Contacting a probe with the hybridizing nucleic acid;
(D) contacting the hybridization nucleic acid sandwich with a transition metal complex that oxidizes the predetermined base by an oxidation-reduction reaction, wherein there is transfer of electrons from the predetermined base to the transition metal complex, and a catalytic cycle A reduced form of the transition metal complex is regenerated as part of
(E) detecting the presence or absence of the redox reaction associated with the hybridizing nucleic acid sandwich;
(F) an object in the test sample presumed to contain the same, comprising the step of determining the presence or absence of the target nucleic acid in the test sample from the detected redox reaction at the predetermined base A method for detecting the presence or absence of a nucleic acid. (A) a conductive substrate having a working surface thereon; (b) a polymer layer is permeable to the transition metal complex, and the polymer layer is connected to the working surface, which binds to the nucleic acid. was composed of a non-conductive polymer layer, Ri by to react the transition metal complex and the nucleic acid capable of oxidizing said predetermined base in an oxidation-reduction reaction, wherein said transition from said predetermined base There is transfer of electrons to the metal complexes, reduced form of the transition metal complex as part of the catalytic cycle is Ru is reproduced, electrodes useful in electrochemical detection of a given nucleotide in the nucleic acid. The nucleic acid comprises at least one predetermined base;
(A) (i) a conductive base plate having an active surface formed thereon; and (ii) a polymer layer that binds to the nucleic acid thereto and is connected to the active surface Contacting a sample containing the nucleic acid with an electrode comprising a conductive polymer layer;
(B) the polymer layer is permeable to the transition metal complex, a nucleic acid, comprising the steps of reacting with the transition metal complex capable of oxidizing said predetermined base in an oxidation-reduction reaction, of the predetermined There is a transfer of electrons from the base to the transition metal complex, and the reduced form of the transition metal complex is regenerated as part of a catalytic cycle ;
(C) detecting the redox reaction by measuring a current flow through the electrode;
(D) A method for detecting a nucleic acid comprising the step of determining the presence or absence of the nucleic acid from the redox reaction detected at the predetermined base.

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