KR19990028484A - Electrochemical detection method of nucleic acid hybrids - Google Patents

Abstract

(A) reacting a nucleic acid with a transition metal complex capable of oxidizing a base preselected by an oxidation-reduction reaction; (b) detecting an oxidation-reduction reaction; (c) measuring one or more pre-selected bases (e.g., adenine, mercapto guanine, 8-oxo-guanine, and 8-oxo (E. G., DNA, RNA) containing the nucleotide sequence (e. This method can be used in many applications, for example, in DNA sequencing, diagnosis and quantitative analysis.

Description

Electrochemical detection method of nucleic acid hybrids

Detecting each DNA sequence in a heterogeneous sample provides the basis for a new approach to gene identification, DNA contouring, and DNA sequencing. One method of detecting DNA hybrids is to use surface-bound DNA sequences that can be verified using analytical sensitivity to hybridization of oligomers surface-bound to a sequence in a heterogeneous sample. These analytical methods generally involve laser-induced fluorescence from labels covalently attached to the target DNA chain, which is not sensitive to single-base mismatches in surface-bound duplexes. See, for example, U.S. Patent Nos. 5,143, 854 and 5,405, 783; Nature 364: 555 (1993); Angew. Chem. 107: 356 (1995); Analytica Chemistry 67 (5): 201A (1995) proposes a surface or " chip " for this application. Biochem. and Molec. Bio. Inter. 32 (1): 21 (1994), DNA hybrids were detected by an electrochemical method involving comparative observation of oxidation-reduction reactions of single-chain DNA and double-stranded DNA. This technique is also not sensitive to single base mismatches in DNA samples. In U.S. Patent No. 5,194, 372 and the like, techniques for detecting single-base mismatches, including enzymatic or chemical degradation, have been proposed. However, such a technique requires a long time and requires a separation technique.

U.S. Patent No. 5,312,527 describes a sequence-selective voltage-current sensor for detecting a target nucleic acid in a double-stranded nucleic acid contacting a complex active in an oxidation-reduction reaction. Since the complex itself is a compound that is active in the oxidation-reduction reaction to provide a voltage-current signal, the complex does not act on the catalytic method.

U.S. Patent No. 4,840, 893 describes an electrochemical verification method for nucleic acids in which competitive binding between a ligand and an anti-ligand is electrochemically detected in turn.

Therefore, there is a need for a method for detecting DNA hybrids, including methods for detecting single-base mismatches that are fast, sensitive, and readily applicable on-line.

The present invention relates to nucleic acid hybrids and sequencing, and more particularly to a method for qualitatively and quantitatively detecting nucleic acid hybrids and a nucleic acid sequencing method.

Figure 1 shows the circulating voltage current of Ru (bpy) ₃ ²⁺ when calf thymus DNA is present and absent. The solid line shows a scan of 50 μM Ru (bpy) ₃ ²⁺ at 25 mV / s in 700 mM NaCl / 50 mM sodium phosphate buffer. The dotted line shows the voltage current of 3.0 mM (nucleotide) calf thymus DNA and 50 μM Ru (bpy) ₃ ²⁺ .

FIG. 2 shows the cyclic voltammetry of Ru (bpy) ₃ ²⁺ when 5'-AAATATAGTATAAAA is present as a single chain (C) and when hybridized with the complementary chain (A & B). (A) represents a fully hybridized DNA (5'-AAATATAGTATAAAA) (3'-TTTATATCATATTTT) double-stranded at 25 μM Ru (bpy) ₃ ²⁺ + 100 μM (at the guanine nucleotide). (B) is a Ru (bpy) ₃ ²⁺ and shows a double-stranded (5'-AAATATAGTATAAAA) · (3' -TTTATATAATATTTT) containing a GA mismatch, (C) is a Ru (bpy) ₃ ²⁺ and a guanine (5'-AAATATAGTATAAAA) containing nucleotides.

Figure 3 shows an example of a device useful for carrying out the method of the present invention.

Figure 4 shows a detection method particularly advantageous for the quantitative detection of DNA in which a pre-selected base is located in a target nucleic acid.

Figure 5 shows the cyclic voltammetry of 25 [mu] M Ru (bpy) ₃ ²⁺ at a scan rate of 25 mV / s in a pH 7, 700 mM NaCl / 50 mM sodium phosphate buffer. (A) No oligonucleotide added. (B) 75 μM d [5'-TTTATACTATATTT] added. (C) 75 μM of oligomer of d [5'-GGGAAATATAGTATAAAAGGG] and oligomer from B was added. Working electrode: tin doped indium oxide. Reference electrode: Ag / AgCl. Corresponding electrode: platinum wire. The secondary structure of the hybrid from C is shown in the figure.

Figure 6 (A) 25μM Ru (bpy) 3 2+, (B) 25μM Ru (bpy) 3 2+ and 0.3mM inosine 5'-mono-phosphine faders agent, and (C) 25μM Ru (bpy) 3 2+ And 0.3 mM guanosine 5'-monophosphate. The structures of inosine and guanosine are shown in the figure.

Figure 7 is an illustration of another embodiment of the invention of Figure 4, wherein the preselected base is on an extension product of an end-transferase.

FIG. 8 is a diagram showing another example of the invention of FIG. 4, which is performed in the sandwich analysis format.

9 is a plan view of a microelectronic device useful in the present invention.

Figure 10 is a side view of a portion of the apparatus shown in Figure 9;

Figure 11 shows the effect of DNA in 200 μM Ru (bpy) ₃ ²⁺ , low salt (no NaCl addition) buffer in DNA-absorbed nylon in 200 μM Ru (bpy) ₃ ²⁺ , high salt (700 mM NaCl addition) buffer in buffer-absorbed nylon - shows cyclic voltage currents using nylon-modified ITO electrodes of 200 [mu] M Ru (bpy) ₃ ²⁺ in absorbed nylon.

Figure 12 shows the cyclic voltammetry of 200 [mu] M Os (bpy) ₃ ²⁺ using nylon-denatured ITO electrodes absorbed in buffer or DNA. 12A shows a circulating voltage current with 700 mM NaCl added. 12B shows a circulating voltage current to which NaCl is not added.

Figure 13 shows the effect of tRNA-absorbed nylon in 200 μM Ru (bpy) ₃ ²⁺ , low salt (no NaCl addition) buffer in 200 μM Ru (bpy) ₃ ²⁺ , high salt (700 mM NaCl addition) buffer in buffer-absorbed nylon, - shows cyclic voltage currents using nylon-modified ITO electrodes of 200 [mu] M Ru (bpy) ₃ ²⁺ in absorbed nylon.

14 shows the cyclic voltammetry with 5'-AAATATAG _n TATAAAA when 25 μM Ru (bpy) ₃ ²⁺ alone and n = 1 (G), 2 (GG) or 3 (GGG) (100 μM). The scan rate is 25 mV / s.

15 shows the cyclic volt- age current with 5'-AAATAT (AGT) _n ATAAAA when 25 μM Ru (bpy) ₃ ²⁺ alone and n = 1 (G), 2 (GG) or 3 (GGG) . The scan rate is 25 mV / s.

Figure 16 is a graph comparing the absorbance of 25 μM ruthenium (4,4'-dimethylbipyridine) ₃ ²⁺ (or "Ru (4,4'-Me ₂ -bpy) ₃ ²⁺ ") alone (Dotted line) and 5'-AAATATAGGGTATAAAA (dashed line) of 100 [micro] M). The scan rate is 25 mV / s.

17 shows the cyclic voltammetry of Ru (4,4'-Me ₂ -bpy) ₃ ²⁺ in 50 mM sodium phosphate buffer with 700 mM NaCl added at a scan rate of 25 mV / s. Curve A shows Ru (4,4'-Me ₂ -bpy) ₃ ²⁺ alone. Curve B shows Ru (4,4'-Me ₂ -bpy) ₃ ²⁺ in the presence of 0.70 mM 6-mercapto guanosine 5'-monophosphate.

18 shows the cyclic voltammetry of 200 [mu] M Ru (bpy) ₃ ²⁺ on the ITO working electrode with Hybond N + nylon membrane attached. The membrane is impregnated with poly [C] and hybridized to the buffer (A) and the poly (G) concentrate (B).

Fig. 19 shows the cyclic voltammetry of 200 [mu] M Ru (bpy) ₃ ²⁺ on an ITO working electrode with Hybond N + nylon membrane attached. The membrane is impregnated with poly [G] and hybridized to the buffer (A) and concentrate (B) of calf thymus DNA.

FIG. 20 shows the cyclic voltammetry of 200 μM Ru (bpy) ₃ ²⁺ on nylon-denatured glassy carbon electrode after absorbing DNA into (A) or nylon film without DNA. The scan rate is 25 mV / s.

The present invention provides a method for detecting a nucleic acid comprising one or more preselected bases (e.g., adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine). The method comprises: (a) reacting the nucleic acid with a transition metal complex capable of oxidizing a preselected base in an oxidation-reduction reaction; (b) detecting the oxidation-reduction reaction; (c) measuring the presence or absence of the nucleic acid from the oxidation-reduction reaction detected in the preselected base. According to a specific example and preferred specific object of the present invention, the present invention can include the step of forming a hybrid nucleic acid by contacting the nucleic acid with a complementary nucleic acid.

First, the present invention provides a method for detecting a DNA hybrid. The method comprises: (a) contacting a DNA sample with an oligonucleotide probe to form a hybrid DNA; (b) reacting the hybrid DNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having at least one preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the presence or absence of the hybrid DNA from the oxidation-reduction reaction detected in the preselected base. As described below, the step of detecting the oxidation-reduction reaction is carried out by measuring the electron flow from the preselected base.

The present invention also provides a method for detecting another DNA hybrid. The method comprises: (a) contacting a DNA sample with an oligonucleotide probe to form a hybrid DNA; (b) reacting the hybrid DNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having at least one preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the detected oxidation-reaction rate; (e) comparing the measured reaction rate with the rate of oxidation-reduction reaction of the transition metal complex and single-chain DNA; And (f) measuring whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the transition metal complex and the single-chain DNA.

The present invention also provides a DNA hybrid detection apparatus. The apparatus comprises: (a) a plurality of DNA sample vessels; (b) sample manipulating means for transferring the plurality of DNA sample containers; (c) oligonucleotide probe transfer means for transferring the oligonucleotide probe to each of the DNA sample containers; (d) transition metal complex transfer means for transferring the transition metal complex to each of the DNA sample containers; And (e) an oxidation-reduction reaction detector for detecting oxidation-reduction reaction.

The present invention also provides another DNA hybrid detection device. (A) a DNA sample container; (b) an oligonucleotide probe transfer means for transferring the oligonucleotide probe to the DNA sample container; (c) transition metal complex transporting means for transporting the transition metal complex to the DNA sample vessel; And (d) an oxidation-reduction reaction detector for detecting oxidation-reduction reaction.

The present invention also includes a method for sequencing DNA. The method comprises: (a) contacting a DNA sample with an oligonucleotide probe comprising a preselected base having a unique oxidation potential to form hybrid DNA; (b) reacting the hybrid DNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having a preselected number of preselected bases in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the reaction rate of the detected oxidation-reduction reaction; And (e) identifying the base pairing with the preselected base.

Hereinafter, the present invention will be described in detail.

As used herein, the term " nucleic acid " refers to a nucleic acid comprising both DNA and RNA. The nucleic acid of the present invention is a polymer of each nucleotide covalently linked by a 3 ', 5' phosphodiester linkage.

As used herein, the term " complementary nucleic acid " refers to a nucleic acid comprising an oligonucleotide probe that specifically binds to another nucleic acid to form a hybrid nucleic acid.

&Quot; Measurement of an anatomical site " includes both qualitative and quantitative measurements of the presence of a detectable substance (e.g., DNA hybrid, RNA hybrid, target nucleic acid detection, etc.).

&Quot; Hybrid DNA " and " hybrid nucleic acid " means single-chain DNA hybridized to form double-stranded DNA or nucleic acid, or double-stranded DNA or nucleic acid hybridized to form triple helix DNA or nucleic acid.

The methods and apparatus of the present invention are sometimes described for DNA for clarity, but may also be applied to other nucleic acids such as RNA.

A. Method of nucleic acid propagation

The method of the present invention includes a step of contacting a DNA sample with an oligonucleotide probe to produce hybrid DNA, and in some cases, it is preferable to amplify the DNA before contacting the probe. The propagation of the selected, or target, nucleic acid sequence is done by appropriate means. Am. Biotechnol. lab. 8, 14-15 (1990). Examples of suitable expression techniques include, but are not limited to, polymerase chain reaction (including reverse transcriptase polymerase chain reaction for RNA amplification), lagase chain reaction, chain substitution proliferation, transcription based proliferation (Proc. Na Acad Sci. USA 86, 1173 (See Biotechnolgy. 6 1179-1202 (1988)), nucleic acid sequences (see, for example, < RTI ID = 0.0 & (See Genetic Engineering News 12 (9), (1992)), chain repair reaction (see Genetic Engineering News 12 (9), 1992), boomerang DNA propagation ). The base bound to the proliferation product is a natural or modified base (pre- or post-proliferation), and the base is selected to optimize subsequent electrochemical detection.

Polymerase chain reaction (PCR) can be performed by known techniques. U.S. Patent No. 4,683,195; 4,683,202, 4,800,159, and 4,965,188. Generally, PCR involves first treating with an oligonucleotide precursor for each chain of a particular sequence that is detected in hybrid conditions (e.g., in the presence of thermostable DNA polymerase) in nucleic acid samples. Thus, the extension product of each precursor is complementarily synthesized to each nucleic acid chain. The elongate products synthesized from each precursor, when separated from the complement, serve as templates for the synthesis of elongation products of different precursors. Thereafter, if the sequences or sequences to be detected are present, the sample is processed under denaturing conditions to separate the precursor elongation product from their template. This step is repeated until a desired degree of propagation is obtained. Detection of the proliferation sequence is performed by adding an oligonucleotide probe capable of hybridization to the reaction product (e.g., an oligonucleotide probe of the present invention) to the reaction product. The probe carries a detectable label and detects the label according to known techniques. When the nucleic acid to be proliferated is RNA, the proliferation is performed according to a known technique by first converting the DNA into a reverse transcriptase.

Chain displacement proliferation (SDA) can be done by well known techniques. Proc. Natl. Acad Sci. USA 89, 392-396 (1992); Nucleic Acids Res. 20, 1691-1696 (1992). For example, SDA is performed by a single proliferation precursor or a pair of proliferative precursors, and exponential growth by the latter. Generally, SDA proliferation is carried out in a 5 'to 3' direction, with a sequence located on the side (DNA sequence is not critical), a restriction site for the restriction enzyme used in the reaction, and a target sequence to be propagated and / Hybridizing oligonucleotide sequences (e. G. Oligonucleotide probes of the invention). The sequence located on the side facilitates binding of the restriction enzyme to the restriction site and provides a DNA polymerase preference site after the restriction site has been labeled, preferably 15 to 20 nucleotides in length; Restriction sites act on the SDA reaction; The oligonucleotide probe site is preferably 13 to 15 nucleotides in length.

Ligase chain reaction (LCR) is also performed by known techniques. Science 254, 1292 (1991). Generally, two pairs of oligonucleotide probes are performed. The pair joining one of the sequences of the detected sequence; The other pair joins the other pair of detected sequences. Each pair together overlaps the corresponding chain completely. In the reaction, first, the sequence chain to be detected is denatured (for example, separated), the pair of oligonucleotide probes are reacted with the chain in the presence of heat-stable ligase to ligate each pair of oligonucleotides to each other, And repeating this process until a desired degree of propagation is obtained. Detection is carried out in the same manner as described for the PCR.

B. Oligonucleotide probe

As described above, the method of the present invention is useful for the detection of DNA hybrids. The first step in the method is to contact the DNA sample with an oligonucleotide probe to form the hybrid DNA. Oligonucleotide probes useful in the methods of the present invention can be composed of between 4 and 6 bases between 80 and 100 bases, more preferably between 8 and 15 bases. Oligonucleotide probes with various types of base sequences are known in the art. Suitable bases for preparing oligonucleotide probes include natural nucleotides such as adenine, cytosine, guanine, uracil and thymine; 2-thioxanthine, 8-oxo-guanine, 6-mercapto guanine, 4-acetylcytidine, 5- (carboxyhydroxyethyl) uridine, 2-O-methylguanosine, 2-O-methylguanosine, inosine, N6-isopentyladenosine, N-isohexyladenosine, Methylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 2-methylguanosine, 2-methyladenosine, 5-methoxyaminomethyl-2-thiouridine,?, D-mannosylquinazines, 5-methylthiourea, 5-methylthioguanidine, (9- [beta] -D-ribofuranosyl-2-methylthiopurin-6-yl) carboxamidine, 5-methoxyuridine, 2-methylthio-N6-isopentyladenosine, N- (9- [beta] -D-ribofuranosyl-6-yl) N-methyl-carbamoyl) threonine, uridine 5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, tootoxin, pseudouridine, quiozine, 2-thiocidin, 5-methyl-2-thiouridine, 2- Methyl-5-uridine, 2'-O-methyl (2-methyl-5-pyridyl) Is an unnatural or synthetic nucleotide base such as uridine, povidosine, and 3- (3-amino-3-carboxypropyl) uridine. Oligonucleotide backbones containing DNA, RNA (although RNA is less desirable than DNA), denatured sugars such as carbocycles, and sugars containing 2 'substituents such as fluoro and methoxy may be used. An oligonucleotide can be an oligonucleotide, wherein the phosphat residues connecting the one or more or all of the nucleotides are modified phosphatides. Modified phosphates include methyl phosphonate, methyl phosphonothioate, phosphoramorpholidate, phosphopiperazidate, and phosphoramidate. The oligonucleotide may be a peptide nucleic acid such as those described in Science 254, 1497-1500 (1991). The only requirement is that the oligonucleotide probe should have a sequence that can bind to the known portion of the DNA sample sequence. In some cases, it is desirable to bring the DNA sample into contact with a plurality of oligonucleotide probes having different sequences (e.g., when there is more than one target nucleic acid in the sample, or in a sandwich assay, a single target nucleic acid is present in more than one probe Hybrid)

C. Hybrid method

The DNA (or nucleic acid) sample is contacted with an oligonucleotide probe by any suitable known method. For example, a DNA sample is dissolved in a solution to dissolve the oligonucleotide probe into a DNA solution under conditions permitting hybrids. Suitable conditions are known (see U.S. 4,358, 535) and include high salt concentration conditions. The DNA sample may be dissolved in a solution of an oligonucleotide immobilized on a solid support to immerse a solid carrier having an oligonucleotide probe immobilized thereon in a solution containing a DNA sample.

D. Oxidizing agents and oxidation-reduction methods

When the hybrid step proceeds to the oxidation step, the hybrid DNA is reacted with a suitable oxidizing agent capable of oxidizing the preselected base in the oxidation-reduction reaction. The preselected base is a natural or synthetic nucleotide in an oligonucleotide probe that undergoes oxidation in reaction with the selected oxidizing agent. Preselected bases exhibit unique oxidation potentials when paired compared to when they are not paired. Preselected bases exhibit unique oxidation potentials when paired with four natural bases. Generally, a 5'-mononucleotide (e.g., a 5'-deoxyribonucleotide or a 5'-ribonucleotide) exhibiting a rate constant of 10 ⁴ M ^-1 S ^-1 or greater can be detected using a catalytic reaction. Examples of the base include guanine, adenine, 8-oxo-guanine, 8-oxo-adenine, 8-bromoguanine, guanosine, xanthosine, thiosin, pseudoridine, 6- Mercaptopurine, 2-thioxanthine, 6-thioxanthine, 6-mercaptopurine, 2-amino-6-carboxymethyl-mercaptopurine, 2-mercaptopurine, 6-methoxypurine, Aminopurine, 6-amino-2-dimethylallyl, 2-aminopurine, 2-aminopurine, 2-thioadenine, 8-hydroxyadenine, 8-methoxyadenine. Generally, the preselected base is guanine, adenine, 6- mercaptojuanine, -Guanine, and 8-oxo-is selected from the group consisting of adenine, guanine and the preferred natural base is a base other than the preferred synthetic 6-mercapto togu.

An oxidizing agent is a charged molecule such as a cation, anion, or amphoteric molecule that is reactive to a preselected base at a unique oxidation potential. Thus, the choice of oxidizing agent depends on the chosen base, which is a skill that is readily available to those skilled in the art. Particularly preferred oxidizing agents include transition metal complexes which are capable of undergoing metal-DNA electron transfer with a preselected base, so as to produce a reduced form of the metal complex to complete the catalytic cycle. Examples of suitable transition metal complexes for use in the process of the present invention are ruthenium ²⁺ (2,2'-bipyridine) ₃ ("Ru (bpy) ₃ ²⁺ "), ruthenium ²⁺ 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⁺ "), Fe ²⁺ _{(2,2'-bipyridine) 3 (" Fe (bpy)} 3 2+ "), Fe ²⁺ (5-chloro-phenanthroline) ₃ (" Fe (5-Cl-phen) ₃ ²⁺ "), osmium ²⁺ (2,2'-bipyridine) ₃ (Os (bpy) ₃ ²⁺ ), osmium ²⁺ (5-chlorophenanthroline) ₃ ("Os (5-Cl-phen) ₃ ²⁺ "), dioxolenium ¹⁺ phosphine, dioxolenium ¹⁺ pyridine ("ReO ₂ (py) ₄ ¹⁺ "). The anionic complexes useful as oxidizing agents are Ru (bpy) ((SO ₃ ) ₂ -bpy) ₂ ^2- , Ru (bpy) ((CO ₂ ) ₂ -bpy) ₂ ^2- and both ionic complexes useful as oxidizing agents Ru (bpy) ₂ ((SO ₃ ) ₂ -bpy) and Ru (bpy) ₂ ((CO ₂ ) ₂ -bpy). (SO ₃ ) ₂ -bpy ^2- is 4,4' ^- disulfonato ^- 2, 2'-bipyridine and (CO ₂ ) ₂ -bpy ^2- is 4,4'-dicarboxy-2 , 2'-bipyridine. Suitable substituted derivatives of pyridine, bipyridine and phenanthroline may also be used for the complexation with the metal. Suitable derivatives include 4-aminopyridine, 4-dimethylpyridine, 4-acetylpyridine, 4-nitropyridine, 4,4'-diamino-2, 2'-bipyridine, Diethylenediamine-2, 2'-bipyridine, 5,6'-diamino-2,2'-bipyridine, 4,4'- 2'-bipyridine, 6'-diethylenediamine-2,2'-bipyridine, 4,4'-dihydroxy-2,2'-bipyridine, 5,5'-dihydroxy- , 2'-bipyridine, 6'-dihydroxy-2,2'-bipyridine, 4,4'4'-triamino-2,2'2'-terpyridine, , 4 "-triethylenediamine-2,2 ', 2" -terpyridine, 4,4', 4 "-trihydroxy-2,2'2" -terpyridine, 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, Dihydroxy-1, 10-phenanthroline, 4, 10-phenanthroline, 3,8-dihydroxy- 7-dinitro-1, 10-phenanthroline, 3,8-dinitro-1, 10-phenanthroline, , 10-phenanthroline, 4,8-disperamine-1, 10-phenanthroline, 3,8- , 2'-c] phenazine.

The oxidizing agent reacts with the hybrid DNA by a suitable method effective for the oxidation-reduction reaction of the pre-selected base and the oxidizing agent. The oxidizing agent should react with the hybrid DNA sample under conditions sufficiently effective for selective oxidation of the preselected base. For example, the transition metal complex can be reacted with an oxidizing agent by dissolving the oxidizing agent in a solution containing the dissolved hybrid DNA under conditions effective for the oxidation-reduction reaction of the preselected base and the oxidizing agent. When the hybrid DNA is immobilized on a solid support, the oxidant is immobilized on the same solid support, and the solid support is immersed in a solution under conditions effective for the oxidation-reduction reaction of the preselected base and the oxidant, ≪ / RTI > The solvent in which the oxidation-reduction reaction takes place can be any solvent that is suitable for dissolving DNA, preferably water.

In hybrid DNA or nucleic acids, the oxidizing agent binds to the small groove of the DNA, thereby eliminating the premature contact of the oxidizing agent with the preselected base by the unique structure of the double (or triple) helix. This protection of the preselected base requires electron tunneling through a solvent, which attenuates the electron transition rate. The solvent accessibility depends on the nature of the nucleotide base paired with the preselected base, and the tunneling distance is estimated to be:

k / _kSs = exp (-? r)

Where r is the distance variation in the double chain compared to the single chain and k _SS is the oxidation rate constant of the base pre-selected in the single-stranded DNA sample. Thus, the tunneling distance between the preselected base and the oxidizing agent depends on each base pair and unpaired DNA. If the driving force for electronic transfer is significantly less than the rearrangement energy (λ), the RTlnk for the modified driving force associated with the approach of the reactant will be linear with a 1/2 slope according to the Marcus theory. Based on the Marcus theory, the absolute rate constant can be calculated by the following equation:

k =? exp [-β (rr ₀ )] exp [- (ΔG + λ) ² / 4RT]

Where r is the rate constant at the diffusion-controlled limit (10 ¹¹ M ^-1 S ^-1 ), r is the distance between the reactant and the product in the activated complex, r ₀ is the closest distance between the reactant and the product , and beta is the influence of the interference medium. As described above, since the preselected base is bound into the DNA, it will impose a limited distance along the electrons that must tunnel the oxidant. Therefore, r and r ₀ are not the same. Β for water is 3 Å ^-1 . This relatively large? Value indicates that a very small change in the tunneling distance results in a significant change in the electron-to-transition rate constant. The DNA structure between the preselected base and the base pairing with the preselected base depends on the base pairing with the preselected base so that the base paired with the preselected base must tunnel between the preselected base and the oxidizer To the tunneling distance. Thus, the relationship between the tunneling distance and the specific base pairing with the preselected base is established.

E. Detection of oxidation-reduction reactions

The occurrence of the oxidation-reduction reaction can be detected by a suitable known technique. For example, the occurrence of an oxidation-reduction reaction can be detected using a detection electrode that senses a change in an electrical signal indicating that an oxidation-reduction reaction takes place. Generally, a detection electrode sensitive to the electron transfer between the oxidizing agent and the hybrid DNA is placed in contact with the solution containing the reacted hybrid DNA and the oxidizing agent. In general, the reference electrode and the auxiliary electrode are also placed in a solution communicating with the detection electrode (most of the current passes through the auxiliary electrode). Suitable detection electrodes are known, for example, glassy carbon electrodes or indium tin oxide electrodes. Likewise, suitable reference electrodes are also known, for example silver / silver chloride electrodes.

Detection of electrical signals associated with oxidation-reduction reactions enables the determination of hybrid DNA. The step of measuring the presence of hybrid DNA generally comprises the steps of (i) measuring the rate of oxidation-reaction, (ii) comparing the known rate of reaction of the single-stranded DNA with the transition metal complex to the measured rate of oxidation- (Iii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate. The reaction rate measuring step is performed by an appropriate means. Relative reaction rates can be measured, for example, by comparing currents as a function of scan speed, probe concentration, target concentration, medium, buffer, temperature, and / or electrochemical method.

The rate of oxidation-reduction reaction can be measured by any suitable technique. In general, the rate of oxidation-reduction reaction can be measured by measuring the electrical signal associated with the oxidation-reduction reaction taking place. For example, the electrical signal associated with the oxidation-reduction reaction can be measured by providing a suitable device that is electrically connected to the detection electrode. For example, a suitable device would be able to measure the electrical signal generated to provide a rate measurement of the oxidation-reduction reaction between the oxidant and the hybrid DNA. Electrical signals are characteristic of electrochemical methods including cyclic voltammetry measurements, normal pulse voltage and current measurements, and square wave voltage and current measurements. Cyclic voltage and current measurement is preferred.

The measured reaction rate is compared to the known reaction rates of single-chain DNA and transition metal complexes. As noted above, the tunneling distance between a selected base and an oxidizing agent in a single-chain or hybrid DNA affects the rate of oxidation-reduction reaction between the oxidizing agent and the preselected base. Thus, hybrid DNA represents a different oxidation-reduction reaction rate than single-stranded DNA. The presence of the hybrid DNA in the preselected base can be measured by measuring whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the oxidant and the preselected base in the single-stranded DNA. Also, since the tunneling distance between the oxidizing agent and the preselected base will depend on the preselected base and the coupling distance of the pair, it is possible to distinguish each possible base pair from the others. The binding distance of the preselected base and the pair depends on the base pairing with the preselected base. For example, the rate of oxidation-reduction reaction for the oxidation of guanine paired with adenine differs from the rate of oxidation-reduction reaction for the oxidation of guanine paired with cytosine, and in turn, the oxidation of guanine - It is different from the rate of reduction reaction and differs from the rate of oxidation-reduction reaction for the oxidation of guanine paired with thymine. More specifically, the rate of oxidation-reduction reaction for the oxidation of guanine is guanine in pairs with guanine> guanine in pair with guanine> guanine in pair with guanine> guanine in pair with thymine> cytosine. Thus, the methods of the present invention are useful for detecting single base pair mismatches in preselected bases or in base pairs adjacent to preselected bases.

Preferably, when paired with each of the various natural bases, the distinction between oxidation-reduction kinetics for oxidation of preselected bases enables the identification of bases paired with preselected bases. Identification of the base pairing with the preselected base can be accomplished by (i) measuring the rate of oxidation-reaction detected, and (ii) determining the rate of oxidation of the DNA with an adenine, cytosine, guanine, or thymine base attached to a preselected base, Comparing the four different known reaction rates with the measured oxidation-reaction rate, and (iii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate. The reaction rate can be measured according to the above-described technique. The reaction rates of each of the transition metal complexes with DNA having an adenine, cytosine, guanine, or thymine base bound to a preselected base can also be measured according to the same technique, and these rates are known. The measured rate of oxidation-reduction reaction of the oxidizing agent and the hybrid DNA is compared with the known reaction rate of the transition metal complex with the DNA having adenine, cytosine, guanine, or thymine base bound to the preselected base. For example, bases paired with preselected bases are measured by measuring known well-known pairings having an oxidation-reduction reaction rate equal to the measured oxidation-reduction reaction rate.

F. DNA sequencing

(A) contacting a DNA sample with an oligonucleotide probe comprising a preselected base having a unique oxidation potential to form hybrid DNA; (b) reacting the hybrid DNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having a preselected number of preselected bases in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the reaction rate of the detected oxidation-reduction reaction; And (e) identifying a base pairing with the preselected base.

As in the above method, the DNA sample can be proliferated prior to contact with the oligonucleotide probe, according to known techniques. The synthetic base is selected from the group consisting of the above bases and other known bases. The only restriction on synthetic bases is that they have inherent oxidation potentials that are distinct compared to the four natural bases: adenine, guanine, cytosine, and thymine. Contacting the DNA sample with an oligonucleotide probe; Reacting the hybrid DNA with an oxidizing agent; Oxidation-reduction reaction is detected. Identification of the base pairing with the preselected base can be accomplished by: (i) detecting four different known reaction rates of the DNA having the adenine, cytosine, guanine, or thymine base bound to the preselected base and the transition metal complex, And (ii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate.

In another example, the oligonucleotide probe may further comprise a second preselected synthetic base. The second preselected synthetic base has a unique oxidation potential different from the first preselected synthetic base. In this example, the detecting step is further comprised of detecting a redox reaction between the transition metal complex and the second preselected base. The measuring step is further comprised of measuring the reaction rate of the oxidation-reduction reaction between the transition metal complex and the second preselected base. The identifying step is further comprised of identifying a base pairing with the second preselected base. According to this example, oxidation-reduction reactions of both pre-selected bases are detected and bases paired with each preselected synthetic base are identified by this method. As will be apparent to those skilled in the art, the method may be performed with two or more preselected bases. However, the oxidation potentials of the synthetic bases should be different from each other, and have different intrinsic oxidation potentials from the oxidation potentials of the four natural bases.

Repeating the steps of the method with a sufficient number of oligonucleotide probes with the preselected synthetic base at a different site identifying each base in the DNA sample as many as the number of bases paired with preselected bases identified in accordance with the present invention DNA sequencing can be performed. In other words, by providing a sufficient number of oligonucleotide probes, each probe sequence comprising one or more preselected synthetic bases, each synthetic base being located at a different calculated site along the probe sequence of each oligonucleotide probe DNA sequencing can be performed. In this method, repeated detection of the oxidation-reduction reaction of synthetic base DNA and oxidant, measurement of the rate of oxidation-reduction reaction, and identification of the base pairing with the preselected synthetic base lead to the identification of the bases of the sequence of the DNA sample.

G. Devices

The present invention also provides an apparatus useful for carrying out the method of the present invention. An example of such a device is shown in Fig. Generally, the apparatus comprises a plurality of DNA sample vessels 10. The driving device 11 serves as a sample processing means for transferring a plurality of DNA sample containers 10. The liquid receiver 12, the feed line 13 and the valve 14 serve as transport means for transporting the oligonucleotide probe to the DNA sample vessel and are connected to the corresponding liquid receiver 15, 17) serve as transferring means for transferring the transition metal complex to each DNA sample container. The probe apparatus 20 includes a drive 21 and a probe 22 and functions as oxidation-reduction reaction detecting means for detecting oxidation-reduction reaction. In operation, the DNA sample is pre-packed in the sample container (10). The driving device 11 moves the sample container 10 below the oligonucleotide probe transfer means and oxidant transfer means. After transfer of each reagent, each sample vessel is moved under the probe 22 by the driving means, and the probe 22 is moved to the sample vessel by the drive 21 to detect the oxidation-reduction reaction. An additional electrode necessary for cyclic voltage and current measurement moves together with the probe 22. The manipulation and data collection of the various components can be performed by a suitable controller 30, such as a program running on a general computer.

Of course, it will be apparent to those skilled in the art that various modifications of the device are possible. Many DNA sample vessels are known well known vessels, such as microtiter plates, test tubes, petri dishes, culture vials, solid supports, etc., which can contain, for example, DNA samples. The sample processing means is a well-known suitable sample processing means capable of moving the DNA sample container.

Suitable oligonucleotide transfer means for transferring oligonucleotides to a DNA sample container are known. For example, in one example, the oligonucleotide probe delivery means comprises a solid support to which the oligonucleotide is immobilized. The oligonucleotide probe transfer means should allow sufficient contact between the DNA sample and the oligonucleotide probe under the appropriate conditions in which the hybridization of the oligonucleotide probe and the DNA is effective. Suitable oxidant transfer means for transferring the oxidant to the DNA sample vessel are known. For example, in one example, the oxidant transfer means comprises a solid support to which the oxidant is immobilized. An oxidation-reduction reaction detector for detecting an oxidation-reduction reaction includes, for example, one or more electrodes capable of detecting oxidation of a preselected base. Suitable sensing and reference electrodes are described in the above reference. Preferably, the electrode is in electrical communication with the means for measuring the reaction rate of the oxidation-reduction reaction. Means for measuring the oxidation-reduction reaction rate are known.

In another example of the present invention, an apparatus for detecting a DNA hybrid comprises (a) a DNA sample container; (b) an oligonucleotide probe transfer means for transferring the oligonucleotide probe to the DNA sample container; (c) transition metal complex transporting means for transporting the transition metal complex to the DNA sample vessel; And (d) an oxidation-reduction reaction detector for detecting oxidation-reduction reaction. This device is described in U.S. Patent Nos. 5,143, 854 and 5,405, 783; Nature 364: 555 (1993); Angew. Chem. 107: 356 (1995); And Analytica Chemistry 67 (5): 21 (1995).

As described above, the DNA sample container is a known suitable container. The oligonucleotide transfer means is preferably a solid support capable of transferring a probe to a DNA sample container and having a plurality of oligonucleotides fixed thereon. For example, a DNA sample in a DNA sample container is contacted with a solid carrier having a plurality of oligonucleotides immobilized thereon under conditions that allow a hybridization of one or more oligonucleotide probes and DNA.

Suitable oxidant transfer means for transferring the oxidant to the DNA sample vessel are described above. A preferred oxidant transfer means comprises a solid carrier to which an oxidant is immobilized. In a preferred embodiment of the present invention, the oligonucleotide probe and the oxidizing agent are immobilized on the same solid carrier.

The apparatus of the present invention is useful for performing diagnostic analysis of various DNA samples. Many oligonucleotide probes enable the detection and analysis of various DNAs in the same container and are useful as screening tools for single samples against various DNA including pathogens, viruses and the like.

H. RNA hybrid detection, RNA sequencing, and RNA mismatch detection

The present invention also provides methods for RNA hybrid detection, RNA sequencing, and RNA mismatch detection. RNAs useful for carrying out such methods include ribosomal RNA, messenger RNA, or genomic RNA (e. G., RNA obtained from RNA viruses such as retrovirus, HIV-1, etc.). According to the present invention, a method for detecting an RNA hybrid comprises: (a) contacting an RNA sample with an oligonucleotide probe to form a hybrid RNA; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having at least one preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the presence of the hybrid RNA from the oxidation-reduction reaction detected in the preselected base.

More specifically, a method of detecting an RNA hybrid comprises: (a) contacting an RNA sample with an oligonucleotide probe to form a hybrid RNA; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having at least one preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the detected oxidation-reaction rate; (e) comparing the measured reaction rate with the rate of oxidation-reduction reaction of the transition metal complex and single-chain RNA; (f) measuring whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the transition metal complex and the single-chain RNA.

The RNA sequencing method comprises: (a) contacting an RNA sample with an oligonucleotide probe comprising a preselected base having a unique oxidation potential to form a hybrid RNA; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing in a redox reaction a preselected base in the nucleotide having a preselected number of preselected bases; (c) detecting the oxidation-reduction reaction; (d) measuring the reaction rate of the detected oxidation-reduction reaction; And (e) identifying the base pairing with the preselected base.

An oligonucleotide probe, a hybrid method, an oxidizing agent, a detection of oxidation-reduction reaction, an apparatus useful for carrying out this method are as described in A-H above, and only RNA is applied according to a principle known as a nucleic acid sample.

I. Detection of preselected bases on the target nucleic acid

In this method, the complexes are used to obtain electrochemical currents from single- or double-stranded DNA or nucleic acids. Pre-selected bases, such as guanine, produce electrochemical signals that are much weaker than double-stranded DNA. Such a method preferably exhibits high sensitivity and can resolve single base mismatches. Thus, such methods are particularly advantageous for DNA sequencing. However, such methods require (a) there is a voice signal going from the probe chain to the hybrid; (b) there is no signal amplification. The following techniques provide solutions to these problems. In addition, the following techniques are particularly useful for diagnostic assays and are particularly useful for quantitative detection of nucleic acids.

The present invention provides a method for detecting a target nucleic acid containing one or more preselected bases in a test sample.

Preferably, the target nucleic acid comprises more than 10 pre-selected bases than the oligonucleotide probe, preferably more than 50 or 100 more pre-selected bases than the oligonucleotide probe. A larger current increase is obtained when the target nucleic acid contains much more pre-selected bases than the oligonucleotide probes.

Optionally, but preferably, the oligonucleotide probes are free from pre-selected bases (i.e., contain a sufficiently small number of preselected bases so as not to interfere with the signal from the probe or to cause errors from the target nucleic acid) . When a natural base that hybridizes to a target nucleic acid can not be used, other bases that are inert to redox are used.

The target nucleic acid is preferably longer than the oligonucleotide probe, and as shown in Figure 4, one or more preselected bases are not hybridized (i.e., overhanging) to the oligonucleotide probe in the hybrid nucleic acid. Preferably, 10, 50, or more than 100 pre-selected bases are over-hanging bases, thereby providing amplification of the detected electrochemical signal.

For example, oligonucleotide probes which do not contain a guanine residue (containing, for example, A, T and C) can be used. The cyclic voltametry of Ru (bpy) ₃ ²⁺ in the presence of this chain is very similar to the absence of oligomers. This chain hybridizes to the target chain containing guanine in either the overlapping region or the overhanging region if the target nucleic acid is longer than the oligonucleotide probe. Since multiple guanines are detected, the signal is amplified relative to the number of hybrids formed. When the genomic DNA or RNA is a target nucleic acid, a large number of overhanging guanines are generated, and a tremendous amplification occurs. For example, ribosomal RNA can contain 1,000 guanines for specific organs, thus providing a 1000-fold amplification per hybrid.

For example, in a preferred example, analysis for preselected bases on the target chain will immobilize the probe chains on a solid surface oriented adjacent to the electrode surface, thereby providing a low background signal when scanned in the presence of the medium. The solid surface is brought into contact with a solution of the target chain, including the preselected base. If a hybrid occurs, the target chain is close to the electrode and an increase in current is detected.

Quantification of nucleic acids.

This method is suitable for quantitative analysis of nucleic acids. In this case, the rate constant for oxidation of the hybrid by the oxidant (for example, Ru (bpy) ₃ ²⁺ ) is measured from the cyclic voltage current (or other electrical signal) by digital simulation. Since this reaction is a secondary reaction, the rate is k [Ru (bpy) ₃ ²⁺ ] [DNA]. Where k is the rate constant, [Ru (bpy) ₃ ²⁺ ] is the concentration of the oxidant, and [DNA] is the concentration of the hybrid (DNA-DNA hybrid). k and [Ru (bpy) ₃ ²⁺ ] are known, the amount of the hybrid can be measured. Obtain a calibration curve for the current increase obtained with a different amount of standard solution containing the target DNA or RNA and obtain the amount of the hybrid directly using the actual increased current. This amount is directly related to the amount of the target substance (eg, the infecting organ in a medical sample). J. Virol. 69, 3510-3516 (1995); Science 272, 1167-1170 (1996).

J. Other bases inert to redox reactions

One disadvantage of the method described in H above is that the oligonucleotide probe does not contain a significant number of preselected bases (e.g., guanine). The solution to this problem is to replace the guanine in the probe chain, but in the presence of other bases which are not oxidized by the oxidizing agent under the reaction conditions (for example, bases having a greater affinity for cytosine than the other bases in the nucleic acid duplex, ). Examples of such bases are inosine and 7-deaza-guanine.

Thus, a method for detecting a target nucleic acid wherein the target nucleic acid contains one or more preselected bases and wherein the probe or the capture nucleic acid comprises a base that is inert to the other oxidation-reduction reactions comprises the steps of (a) contacting the test sample with a target nucleic acid Lt; / RTI > to form a hybrid nucleic acid; (b) contacting the hybrid nucleic acid with a transition metal complex which oxidizes the preselected base in an oxidation-reduction reaction; (c) detecting the presence of an oxidation-reduction reaction associated with the hybrid nucleic acid; And (d) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. Wherein the preselected base in the target nucleic acid is guanine; The target nucleic acid comprises cytosine (which is generally associated with guanine in a complementary nucleic acid); The oligonucleotide probe comprises a corresponding base which binds to cytosine in the hybrid nucleic acid. The other base is selected from the group consisting of inosine and 7-deaza-guanine. The reaction step consists of reacting the transition metal complex with the nucleic acid under conditions effective to oxidize the preselected base without oxidizing the other base.

Useful devices for oligonucleotide probes, hybrid methods, oxidizing agents and oxidation-reduction reaction methods, detection of oxidation-reduction reactions, and performance of the method are described in A-I above.

K. Polymerization of terminally transferase and pre-selected bases

The method described in H above prolongs the target nucleic acid with a terminal transferase to provide a pre-selected base added thereto. As shown in Fig. 7, such a method comprises: (a) contacting a test sample with an oligonucleotide probe, which specifically binds to a target nucleic acid and has a terminal blocked by a terminal transferase to form a hybrid nucleic acid; (b) contacting the hybrid nucleic acid with a solution containing a base selected in advance in the presence of a terminal transferase to produce an extension product consisting of a preselected base; (c) contacting the oligonucleotide probe with a transition metal complex that oxidizes the preselected base in a redox reaction; (d) detecting the presence of the target nucleic acid in an oxidation-reduction reaction; And (e) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. The test sample is preferably separated from the oligonucleotide probe before the detection step, more preferably between steps (a) and (b). The separation can be done using a fixed probe. Alternatively, as described in H above, the probe may be provided as a solution.

Oligonucleotide probes, hybrid methods, oxidizing agents and oxidation-reduction reaction methods, detection of oxidation-reduction reactions, and apparatus useful for the method are described in A-I above.

L. Sandwich Analysis

Another example of the method described in H above is the so-called " sandwich " analysis, shown in FIG. In a sandwich assay, the target nucleic acid is part of three hybrids composed of a capture probe, a target nucleic acid, and a signal probe.

A method of detecting a target nucleic acid in a test sample comprises the steps of: (a) providing a capture probe that specifically binds to the target nucleic acid; (b) contacting the test sample with the capture probe to form a hybrid nucleic acid; (c) contacting the oligonucleotide signal probe, which specifically binds to the target nucleic acid and contains one or more preselected bases, to the hybrid nucleic acid to generate a hybrid nucleic acid sandwich; (d) contacting the hybrid nucleic acid sandwich with a transition metal complex that oxidizes the preselected base in an oxidation-reduction reaction; (e) detecting the presence of an oxidation-reduction reaction associated with the hybrid nucleic acid sandwich; And (f) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. The test sample is preferably separated from the acquisition probe and the separation step occurs between steps (b) and (c) above or between steps (c) and (d). Depending on the type of analysis (homogeneous or heterogeneous), the oligonucleotide capture probe may be immobilized on a solid support (e.g., the interior surface of a polymer particle, plate, or microtiter plate well) Other means may be used.

Several types of sandwiches are known. Selection of the analysis format is not important, and a format suitable for the present invention may be used. For example, an oligonucleotide capture probe can be immobilized on a solid support, as described in U.S. Patent No. 4,486,539. The oligonucleotide probes may comprise polymeric units as described in U.S. Patent No. 4,868,104, and the hybrid nucleic acid sandwiches are separated by polymerization. The signal probe is described in Clinical Chem. 39, 725-726 (1993). A medium polynucleotide in which an oligonucleotide capture probe bound to a fixed polynucleotide described in U.S. Patent No. 4,751,177 may be used. The oligonucleotide probe can be conjugated to a hybrid nucleic acid sandwich separated from the test sample (e.g., biotin) by means of a second member interaction with one member of the specific binding pair, and the other member of the binding pair, (E.g., avidin). See European Patent Application Nos. 0 238 332, 0 139 489 and 0 192 168.

Oligonucleotide probes, hybrid methods, oxidizing agents and oxidation-reduction reaction methods, detection of oxidation-reduction reactions, and apparatus useful for the method are described in A-K above.

M. Background Detection of preselected bases in the presence of guanine signal

The presence of a preselected base in an oligonucleotide probe can also be detected in the presence of a background signal product resulting from the oxidation of guanine. Detection of the mismatch depends on the ability to detect a pre-selected base in an oligonucleotide probe in the presence of four natural bases. Therefore, preselected bases should be able to be oxidized faster than the other four bases.

The present invention provides oligonucleotide probes useful for electrochemical detection of preselected bases in the presence of background guanine signals. The oligonucleotide probe may be an oligonucleotide probe as described in section B above, comprising at least one purine substituent of formula (I): < EMI ID =

The oligonucleotide probe may contain a desired number of bases (for example, 1, 2 or 3 to 5, 10, or 15 or more) according to the binding partner of the base of the above formula. Specific examples of such oligonucleotide probes, and nucleotides useful in the production thereof, include the following compounds of formula (II): < EMI ID =

Wherein:

R ₁ is HO-P (O) (OH) -O-, a nucleotide, or oligonucleotide;

R ₂ is -H, a nucleotide, or an oligonucleotide;

R ₃ is -H, -OH, halogen (fluoro, chloro), alkoxy, amino, or azido; ego;

R ₄ is -O- or CH ₂ -.

It is apparent to those skilled in the art that the oligonucleotide probes described in connection with the above formulas (I) and (II) are prepared by known techniques, and that modifications other than the following examples are possible.

A preferred example of the compound of the formula (II) is that R ₁ is OH-P (O) (OH) -O-. Another preferred example of the compound of formula (I) is that R is H. When R < _{1 >} is a nucleotide or oligonucleotide, the phosphodiester linkage is at the 3 ' end. When R ₂ is a nucleotide or oligonucleotide, the phosphodiester linkage is at the 5 'end.

The compound of formula (I) is preferably included as a base in an oligonucleotide probe which can be used in the method of the present invention, as described in section A-M above. Oligonucleotide probes may, of course, include various bases, but when the oligonucleotide probes are used for electrochemical detection of preselected bases in the presence of background guanine signals, they should include one or more bases of formula (I).

The oligonucleotide probes are 5, 10 or 50 bases or less in length. A specific example of the compound of formula (II) is 6-mercapto guanosine 5'-monophosphate.

N. Electrode Structure

Electrodes useful for electrochemical detection of a base preselected in nucleic acids according to the above method include: (a) a conductive substrate having a work surface; And (b) a non-conducting polymer layer coupled to the working surface and coupled to the nucleic acid. The polymeric layer is a layer that is conjugated to the nucleic acid (e. G., By hydrophobic action or other suitable bonding technique) and is porous to the transition metal complex (i. E., The transition metal can be transferred to the nucleic acid bound to the polymer). The conductive substrate can be a metallic or non-metallic substrate, including a semiconductor substrate (e.g., gold, glassy carbon, indium-doped tin oxide). The conductive substrate may have various physical forms, such as a long probe with a working surface at its end, or a flat sheet with a working surface on its side. The polymer layer can be connected to the work surface by suitable means, for example, clamping the polymer layer to the work surface, evaporating the polymer solution onto the electrode, or electropolymerization. Examples of polymers include nylon, nitrocellulose, polystyrene, and poly (vinylpyridine). The thickness of the polymer layer is not critical, but may be from 100 ANGSTROM to 1, 10, or 100 microns. The electrode is used in all the methods of A-M above. Thus, in general, the invention provides a method of detecting a nucleic acid comprising one or more preselected bases, said method comprising the steps of: (a) contacting a sample comprising a nucleic acid with (i) a conductive substrate having a working surface; And (ii) a non-conducting polymer layer connected to the working surface and bonded to a nucleic acid; (b) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in a redox reaction and wherein the polymer layer is pore-free therewith; (c) detecting the oxidation-reduction reaction by measuring a current flowing through the electrode; And (d) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base.

O. Microelectronic devices

The advantages of the above technique can be achieved by a microelectronic device. A microelectronic device useful in the detection of nucleic acid species in the method comprises a microelectronic substrate having first and second opposing faces; A conductive electrode on the first side; And an oligonucleotide capture probe immobilized on the first surface adjacent to the conductive electrode. The capture probe is placed in close proximity to the adjacent electrode (e.g., 0.1, 1, or 2 to 50, 100, 500, or 1000 microns) so that the redox reaction that occurs in the target nucleic acid that hybridizes to the probe or probe is detected .

9 and 10, the microelectronic device 20 includes a plurality of discrete electrodes 21 on a first opposing face, and a plurality of discrete electrodes 22, And a nucleotide capture probe 22. By providing different, multiple, separate oligonucleotide capture probes, each associated with an electrode, a single compact device is provided that can detect several different hybrids. Each electrode is electrically connected to an appropriate contact 23 to operate in association with the necessary electrical device to perform the detection and measurement steps of the method.

The nucleic acid is immobilized at an appropriate position on the microelectronic substrate by known techniques. See U.S. Patent No. 5,405, 783. The microelectronic substrate may be a semiconductor (e.g., silicon) or a non-semiconductor material (e.g., glass) that can be processed using conventional microelectronic techniques. The electrode is a metal, or a non-metallic material such as polycrystalline silicon. The electrodes can be fabricated using conventional microelectronic techniques, such as dip etch. A variety of suitable microelectronic structures and fabrication techniques are known. VLSI Technology (1983); See VLSI Fabrication Principles (1983).

The present invention will be described in more detail by the following examples. In this embodiment, ㎠ / s is the square centimeter per second, M is the molar concentration, M ^-1 S ^-1 is the mole per second, eV is the electron volt, V is the bolt, nm is the nanometer, GMP is guanosine 5'- Phosphite, and ITO refers to a tin-doped indium oxide electrode. The cyclic volt-ampere was collected with EG + G Princeton Applied Research Potentiostat / Galvanostat, Model 273A, according to the known art. The ITO electrode was processed by processing an ITO-coated soda ash glass sheet available from Delta Technology, Steelwater, Minn. The nylon film used was a HYBOND-N + nylon membrane available from Amersham Corp. of Alington Heights, Illinois, USA.

[Example 1]

Measurement of cyclic voltammogram of Ru (bpy) ₃ ²⁺

Figure 1 shows the circulating voltage current of Ru (bpy) ₃ ²⁺ when calf thymus DNA is present and absent. Catalytic increase is caused by the oxidation of DNA by the oxidized form of the metal complex observed during the single voltage current sweep. The voltage current of the DNA coupled redox couple is much smaller than that of the metal complex (8.0 × 10 ^-6 ㎠ / s) because the diffusion coefficient of DNA (2.0 × 10 ^-7 ㎠ / s) ≪ / RTI > This phenomenon sharply reduces the combined form of current. However, at a sufficiently high ionic strength ([Na ⁺ ] = 0.8M), the binding of the metal complex is too weak to affect the current response. In this case, the current can be analyzed by the EC 'mechanism of the sample.

Ru (bpy) ₃ ²⁺ ? Ru (bpy) ₃ ³⁺ (E)

Ru (bpy) ₃ ³⁺ + DNA → Ru (bpy) ₃ ²⁺ + DNA _ox (C ')

[Example 2]

Analysis of cyclic voltage current

The cyclic voltage current was analyzed using a DIGISIM ^TM data analysis package, fitting the complete current-potential curve to the extracted background. Input variables are E _1/2 for metal complexes and diffusion coefficients for metal complexes and DNA. Therefore, the obtained single variable was the second-order rate constant for Equation 2, k = 9.0 × 10 ³ M ^-1 S ^-1 . The same rate constant was obtained at several scan rates.

The rate constants for the oxidation of DNA by Ru (bpy) ₃ ³⁺ were confirmed by two separate experiments. First, k _obs for Equation 2 was obtained with the COOL ^TM algorithm using a square wave voltage current. The COOL ^TM algorithm uses a different approach from DIGISIM ^TM , but the k _obs for DNA was linear and the secondary rate constant k = 8.2 × 10 ³ M ^-1 S ^-1 . This was in agreement with that obtained with DIGISIM ^TM . Second, Ru (bpy) ₃ ³⁺ samples were prepared and reacted directly with DNA in fast scanning rectification. The time-dependent spectrum between 350 and 600 nm was that Ru (bpy) ₃ ³⁺ was completely converted to Ru (bpy) ₃ ²⁺ without an intermediate and the rate constant k = 1.2 × 10 ³ M ^-1 S ^-1 . Thus, the rate constants for the oxidation of DNA by Ru (bpy) ₃ ³⁺ were finally completed with two independent electrochemical measurements and non-electrochemical rectification techniques measured in completely different ways.

[Example 3]

Analysis of cyclic voltage current

If the driving force for the electron transition is significantly less than the rearrangement energy (λ), the plot of RTlnk vs. drive force (when modified with respect to reactant access) should be a straight line with a 1/2 slope. The rate constants for the oxidation of DNA by various metal (bpy) _{3 &lt}; ₃ ⁺ > derivatives with different redox potentials are given in Table 1 below.

Since the Marcus theory describes the driving force depending on the electron-to-transition velocity, the absolute velocity constant can be analyzed by the following equation:

k =? exp [-β (rr ₀ )] exp [- (ΔG + λ) ² / 4RT]

The rate constants for the oxidation of DNA are listed in Table 1 below.

Where r is the rate constant at the diffusion-controlled limit (10 ¹¹ M ^-1 S ^-1 ), r is the distance between the reactant and the product in the activated complex, r ₀ is the closest distance between the reactant and the product , and beta is the influence of the interference medium. When guanine ions are coupled into DNA, it will impose a limited distance along the electrons that must tunnel the oxidant. Therefore, r and r ₀ are not the same. However, if an electron donor guanosine 5'-monophosphate (GMP) is used, a direct collision of guanine with a metal complex is possible (r = r ₀ ). For Fe (bpy) ₃ ³⁺ and GMP, the rate constant measured by rectification is 2.6 x 10 ³ M ^-1 S ^-1 . The known lambda value for the relevant reaction is 1-1.5 eV, and DELTA G (guanine ^{+ / 0 + / 0} ) for the guanine couple is 1.1 +/- 0.1V.

[Table 1]

Rate constants of guanine oxidation in DNA oligomers by Ru (bpy) ₃ ²⁺

a: The DNA concentration used in the rate constant measurement is based on guanine nucleotides.

b: Measuring distance of tunneling through solvent. The distance calculated according to k / k _ss = exp [-βΔr] dp when β (H ₂ O) = 3 Å and k _ss = 1.8 × 10 ⁵ M ^-1 S ^-1 .

c: Since the rate constant is relative to the guanine concentration, the observed rate for GG mismatch is relatively normalized to other oligomers containing a single guanine.

Figure 2 shows the cyclic voltammetry of (A) Ru (bpy) ₃ ²⁺ when 5'-AAATATAGTATAAAA 5 is present as a single chain (C) and hybridized with the complementary chain. The GMP with r = r ₀ for the single chain and the rate constant k = 1.8 × 10 ⁵ M ^-1 S ^-1 is ΔG (guanine ^{+ / 0} ) = 1.1 V and the λ value is 1.1 eV. This is consistent with the value obtained from guanine oxidation. While there was a sharp increase in the single chain, only a slight increase was observed at the fully hybridized double chain at this scan rate, resulting in a fourfold reduction in hybrid current. Metal complexes such as Ru (bpy) ₃ ²⁺ are known to bind to small grooves in DNA. Therefore, the rate constant for oxidation of the double chain from the distance between the guanine residue and the complex bound to the surface should be 150 times slower. When a metal complex is docked in a small groove, guanine and the metal complex can not make close contact, and the electrons must tunnel through a solvent that separates the guanine residue from the metal complex. Tunneling through water is less effective than through a nonpolar solvent, and the value of β for water is estimated to be 3 Å. Thus, the tunneling distance is calculated by the following equation:

k / k _ss = exp (-? r)

Where? R is the change in distance in the double chain compared to the single chain. The Δr of the fully hybridized double chain is 1.8.

The large β value for water indicates that a small change in the tunneling distance results in a significant change in the electron-transfer rate constant, again reflecting the small disturbance in the DNA structure. Figure 2 shows the cyclic voltammetry of Ru (bpy) ₃ ^{2+ in} the presence of the same double chain with GC base pairs replaced by GA mismatches. The insertion of a GA match results in a doubled current compared to the original double chain, which results in a 16-fold increase in the rate constant (k _GA = 1.9 × 10 ⁴ M ^-1 S ^-1 ). The data for single chain, fully hybridized double chain, and all three GX mismatches are shown in Table 1. The tunneling distance Δr calculated relative to the single chain is also shown. As expected, in the G-purine base mismatch, guanine is more accessible to metal complexes than GT mismatches in which two bases are bound by two hydrogen bonds. Nevertheless, GT mismatch causes four times the rate constant and is easily detectable. Therefore, the oxidation rate constant is in order of guanine (single chain)>GA>GG>GT> GC. The ability to distinguish each of these mismatches provides a basis for mismatch-sensitive detection of the hybrid, which is also sensitive to single base pair mismatches in base pairs adjacent to the preselected base.

[Example 4]

A base denatured to avoid oxidation in the probe chain; Substitution of inosine for guanine

Cyclic voltage currents were collected using an indium tin oxide (ITO) working electrode (area = 0.32 cm 2). Figure 5 shows the cyclic voltammetry of a sample containing 25 [mu] M Ru (bpy) ₃ ²⁺ at a scan rate of 25 mV / s in a pH 7, 700 mM NaCl / 50 mM sodium phosphate buffer. Figure 6 shows the results of a 25 mV / s scan of 25 uM Ru (bpy) ₃ ²⁺ and 0.3 mM inosine 5'-monophosphate or 0.3 mM guanosine 5'-monophosphate dissolved in 700 mM NaCl / 50 mM sodium phosphate buffer It represents the cyclic voltage current at the speed. The scan of mononucleotides without Ru (bpy) ₃ ²⁺ did not show an oxidation current. A cleaned ITO electrode is used for each experiment and the background scan of the buffer alone is excluded from subsequent scans. The secondary guanine oxidation kinetics constant is measured using a DIGISIM ^TM data analysis package, fitting the cyclic voltammetric data to a two-step mechanism. All parameters other than the oxidation reaction are measured from the voltage current of the metal complex alone on the same electrode. The inosine 5'-monophosphate was purchased from Sigma, and the guanosine 5'-monophosphate was purchased from Biochemical, USA, both of which were used without further purification. Oligonucleotides were prepared in UNC Department Pathology and passed through a 3000-molecular weight separation filter to remove mononucleotides. Purified by reverse phase HPLC. CRC Handbook of Biochemistry and Molecular Biology; 1975; The concentration was measured from the light absorption at 260 nm as described in Vol 1. The complementary chain was heated at 90 占 폚 for 5 minutes and slowly cooled to 25 占 폚 over 2 hours to prepare the hybrid of Fig.

This data shows that inosine substituted for guanine in the probe chain provides a probe chain that is inactive for the redox reaction.

[Example 5]

A denatured base to avoid oxidation in the probe chain: 7-deaza-guanine

This example was carried out in the same manner as in Example 4, except that 7-deaza-guanine was used as a base substituted with guanine and inert to the redox reaction to provide a probe chain.

7-deaza-guanine is only oxidized at a rate of 10 ³ M ^-1 S ^-1 . This is slow enough to provide a probe chain that is two orders of magnitude slower than guanine and inert to redox reactions.

[Example 6]

Detection using calf thymus DNA bound to nylon membrane attached to ITO electrode

The nylon film was cut into a circular shape having a diameter of 6 mm, sandwiched in an electrochemical cell, and covered with the ITO electrode portion exposed to the solution.

For experiments in which only the cyclic voltage current of the metal complex is obtained, the ITO electrode is controlled with a buffer solution. A nylon disk (no DNA) is inserted into the electrochemical cell and 200 μM of 200 μM metal complex solution is added. For Os (bpy) ₃ ²⁺ experiments, an equilibration time of 6 minutes is used before the electrochemical analysis. For Ru (bpy) ₃ ²⁺ experiments, an equilibration time of 15 minutes is used before the electrochemical analysis. The cyclic voltage current is collected using a PAR 273A at a scan rate of 25 mV / s.

For DNA experiments, the DNA-absorbed nylon disk is adjusted in an appropriate buffer, then inserted into an electrochemical cell and 200 μM of a 200 μM metal complex solution is added. The cyclic voltammetry is measured at a scan rate of 25 mV / s after a reasonable equilibrium time (6 min for Os (bpy) ₃ ²⁺ and 15 min for Ru (bpy) ₃ ²⁺ ). The DNA-absorbed nylon disk is absorbed in a solution of 5.8 mM calf thymus DNA dissolved in water for about 5 minutes. Various absorption times of 5 minutes to 18 hours were observed. Because DNA is rapidly associated with nylon, short absorption times are commonly used. Under low salt conditions, 50 mM sodium-phosphate buffer is used (pH = 6.8, [Na ⁺ ] = 80 mM). Under high salt conditions, 50 mM sodium-phosphate buffer and 700 mM NaCl solution are used (pH = 6.8, [Na +] = 780 mM). The cyclic voltage current of Ru (bpy) ₃ ²⁺ in the nylon-ITO electrode is shown in Fig. The dashed line shows the voltage current when the nylon membrane is absorbed into the solution of calf thymus DNA before it attaches to the electrode. There is a large catalytic current for DNA-labeled membranes parallel to that observed in solution. This experiment shows that Ru (bpy) ₃ ²⁺ diffuses freely into the nylon film and diffusion of DNA is not necessary to realize the catalytic current. Figure 11 also shows the large catalytic currents observed at low salt concentrations due to the interaction between the medium and the immobilized DNA.

Figure 12A shows the same experiment using Os (bpy) ₃ ²⁺ as the medium. Since the osmium complex does not oxidize guanine, the current increase observed in the presence of guanine should arise from the concentration of the medium due to DNA binding. In fact, the current for Os (bpy) ₃ ²⁺ in the presence of DNA in nylon electrodes is lower than in the absence of DNA. Experiments show that when the DNA binds to the nylon electrode, the increased current for Os (bpy) ₃ ²⁺ is due only to a proprietary catalytic reaction and not due to binding differences. The effect of the salt concentration is shown in Figure 12B, which is small compared to the high salt effect observed for the catalytic reaction.

By binding the DNA to the nylon membrane attached to the ITO electrode, it can be seen that the DNA is detected even when the medium is diffused without spreading the DNA. Thus, detection of DNA is possible if the immobilized probe is sufficiently close to the electrode that a probe-target hybrid is present in the diffusion layer of the medium.

[Example 7]

Detection of RNA bound to nylon membrane attached to ITO electrode

The same procedure as in Example 6 was carried out except that the tRNA of Bakers Yeast purchased from Sigma was used instead of calf thymus DNA. A nylon film disk was absorbed into the tRNA solution as described in Example 6. The circulating voltage current in the presence of Ru (bpy) ₃ ²⁺ is shown in Fig. Catalytic currents were observed in both buffers and more in low salt. The difference in current between the low salt and the high salt was not as remarkable as in the DNA of Example 6. Since tRNA does not bind to cations and DNA, the effect of salts is not remarkable.

The results in Figure 13 show that both RNA and DNA contain guanine, so that RNA can be detected in the same way as DNA. Therefore, the chemical composition of the sugar phosphate backbone does not affect the catalytic current. As a result of this observation, single- and double-stranded DNAs and RNAs comprising DNA and DNA hybrids and denatured backbones such as PNA, carbocycle, phosphorothioate or other substituted ribose, Can be detected.

[Example 8]

Detection of RNA

For quantitative analysis of RNA, DNA (or RNA, PNA, or other backbone) probes are immobilized on a solid support. The probe is denatured to an inactive state by redox by substituting guanine with inosine or 7-deaza-guanine. The immobilized probe is then contacted with a solution of the target RNA (for example, HIV or hepatitis C virus). The solid surface includes immobilized probes that are inert to oxidation-reduction and hybridized to the RNA chain. The solid surface is contacted with Ru (bpy) ₃ ^{2 +} solution and the cyclic volt-ampere of the medium is measured. The catalytic current signals, hybrids, and currents are used to quantify bound RNA chains based on guanine numbers known in the chain.

For detection of RNA mismatches, DNA (or RNA, PNA, or other backbone) probes are immobilized on a solid support. Preselected bases in the probe chain are more readily oxidized than other bases. The surface is contacted with the target RNA solution and contacted again with Ru (bpy) ₃ ²⁺ solution or other medium. The degree of hybridization (complete agreement) is measured in preselected bases in the same manner as DNA.

[Example 9]

Detection of pre-selected base sequences

Is carried out by the method described in Example 3. Figure 14 shows that the current due to 5'-G is the smallest, followed by the current due to 5'-GG, and then the current due to 5'-GGG. This abrupt increase is observed in both single and double chains containing GG and GGG sequences. The increase in current is not simply due to an increase in the number of G's. Thus, as shown in Figure 15, when G is scattered, the current increase due to the addition of G to the same chain is much lower. Since the 5'-G of GGG is much more readily oxidized than a single G, it is possible to select a medium (with a lower oxidation-reduction potential) capable of oxidizing GGG other than G.

16, the Ru (4,4'-Me ₂ -bpy) oligonucleotide single G for cyclic voltammetry of the ^{2 +} ₃ oligonucleotide and the GGG indicates repeated along the scan in the presence of nucleotides. As shown, the catalytic current was only observed in the presence of GGG oligonucleotides. This example shows the ability to regulate the potential of the medium so that a sequence that is more easily oxidized in the presence of background guanine is detected. The same principle can be applied to the detection of a single synthetic base which is more easily oxidized than guanine.

The example shows that it is possible to lower the potential of the medium and distinguish the base or base sequence to be oxidized more easily.

[Example 10]

Background Detection of preselected guanine derivatives in the presence of natural guanine

The disodium salt of 6-mercapto guanosine 5'-monophosphate (6-S-GMP) was prepared by the phosphorylation of 6-mercapto guanosine purchased from Sigma.

Phosphorylation is described by Bull. Chem. Soc. Jpn. 42: According to 3505 (1969) was performed using POCl _3. The disodium salt of 6-S-GMP is purified by HPLC before measuring the voltage and current. The cyclic voltammogram is carried out at high ionic strength, such as in the inosine-5'-monophosphate. The working electrode is an ITO electrode that prevents direct oxidation of 6-S-GMP by attaching a Hybond N + nylon film to the surface. The counter electrode is a platinum wire. The reference electrode is Ag / AgCl. The scan rate is 25 mV / s.

The measured cyclic voltage current is shown in Fig. Curve A shows Ru (4,4'-Me ₂ -bpy) ₃ ²⁺ alone. Addition of 5'-GMP did not increase the wavelength of Ru (4,4'-Me ₂ -bpy) ₃ ²⁺ . However, as shown in curve (B), the addition of 6-mercapto guanosine 5'-monophosphate leads to a sharp current increase. The current peak at the time of addition of 5'-GMP was the same as the curve A. This data shows that it is possible to detect 6-mercapto guanosine base in the presence of background natural base.

[Example 11]

Detection of pre-selected bases and DNA hybrids on the target chain

Hybond N + nylon membrane (Amersham, 480-600 ㎍ / ㎠) is cut into a circle 6 mm in diameter. A nylon disc is placed in a concentrated aqueous solution of poly-cytidyl acid (Sigma) and absorbed for 1 hour. The disc is removed from the poly-cytidyl acid (poly [C]) solution and placed on the parafilm and dried. After drying, 15 μl of poly [C] solution is added to the film by dividing into 3 times by 5 μl. The disk is completely dried and washed with low salt buffer (50 mM sodium-phosphate, pH = 6.8, [Na ⁺ ] = 80 mM) to remove strongly unbound poly [C].

As a control experiment, poly [C] impregnated discs undergo a hybridization process that exposes them to other hybridization steps without exposing them to nucleic acids. The disk was placed in 400 占 퐇 of Milli-Q water, heated at 48 占 폚 for 1 hour, and then cooled at room temperature. The disk is removed from the water and washed with a low salt buffer for electrochemical analysis. The disc manufactured by this method shows (A) in Figs. 18 and 19.

The poly [C] impregnated disk was placed in an aqueous solution of poly (phthalic acid) (manufactured by Sigma Co.) at 400 어, heated at 48 캜 for 1 hour, and then cooled at room temperature. The disk is removed from the water and washed with a low salt buffer for electrochemical analysis.

Calf thymus DNA in water is denatured by heating at 90 ° C for 1 hour. The poly [C] impregnated disk is placed in denatured calf thymus DNA solution, heated at 48 ° C for 1 hour, and then cooled at room temperature. The disk is removed from the water and washed with a low salt buffer for electrochemical analysis. As a control experiment, a disk not impregnated with poly [C] is subjected to the same process. Binding and detection of calf thymus DNA by absorption with nylon film is observed in the control group.

The ITO electrode is cut with a low salt buffer and the treated nylon disk is inserted into the electrochemical cell as described above. 200 [mu] l of Ru (bpy) _{3 <} ^{2 +} > is added to the electrochemical cell and exhibits a cyclic voltage current after an equilibration time of 15 minutes. The scan rate is 25 mV / s.

The cyclic voltage current is shown in Fig. The probe sequence poly [C] is immobilized on the Hybond N + nylon membrane and the hybrid is carried out in the buffer. The film is attached to the ITO electrode and a circulating voltage current of Ru (bpy) ₃ ²⁺ is obtained (A). The membrane is immersed in poly [G] and the hybrid is performed according to the same method. The cyclic voltametry of Ru (bpy) ₃ ²⁺ is measured (B) and an increase in large current due to the catalytic oxidation of the hybridized poly [G] target is obtained. As shown in Fig. 19, the analysis is specific to the appropriate sequence. Figure 19 compares the cyclic volt- ages of the membranes of poly [C] when the hybrid is performed in buffer (A) or in single-chain calf thymus DNA solution. Figure 19 shows that there is no current increase if no target sequence is present.

[Example 12]

Detection of DNA in nylon-modified glassy carbon electrodes

20 shows the cyclic voltammetry of the glassy carbon electrode with the nylon film adhered to the (A) and (B) nylon films by fixing the DNA to the nylon film.

Nylon membrane (Zeta Probe, Bio-Rad, 80-100 占 퐂 / cm2) was cut into a circle having a diameter of 5 mm. Cover the surface of the glassy carbon electrode with a nylon disc and fix it with a plastic sleeve. In experiments where only the CV of metal complexes was obtained, the glassy carbon electrode was first controlled with a low salt buffer (50 mM Sodium Phosphate, pH = 6.8, [Na ⁺ ] = 80 mM). A nylon disk was attached to the electrode and 400 μl of 200 μM Ru (bpy) ₃ ²⁺ was added to the electrochemical cell. After 15 min equilibration, the circulating voltage current was measured at a scan rate of 25 mV / s.

The above embodiments are for illustrating the present invention, and the present invention is not limited thereto. It will be apparent to those skilled in the art that variations and modifications within the scope of the appended claims are possible.

Claims (151)

(a) contacting a DNA sample with an oligonucleotide probe to form hybrid DNA; (b) reacting the hybrid DNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having at least one preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the presence or absence of the hybrid DNA from the oxidation-reduction reaction detected in the preselected base. 2. The method of claim 1, wherein the measuring step comprises the steps of: (i) measuring the detected oxidation-kinetics rate; and (ii) comparing the measured kinetics with the rate of oxidation-reduction reaction of the transition metal complex and single- , And (iii) measuring whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the transition metal complex and single-chain DNA. The method according to claim 1, wherein the DNA sample is a single-stranded DNA sample, and the hybrid DNA is a double-stranded DNA. 2. The method of claim 1, wherein the oligonucleotide probe comprises 4 to 100 bases. 2. The method of claim 1, wherein the preselected base is guanine. 2. The method of claim 1, wherein the preselected base is adenine. The method of claim 1, wherein the transition metal complex is ^{_{Ru (bpy) 3 2+, Ru}} (Me 2 -bpy) 3 2+, Ru (Me 2 -phen) 3 2+, Fe (bpy) 3 2+, Fe (5-Cl-phen) ₃ ²⁺ , Os (5-Cl-phen) ₃ ²⁺ , and ReO ₂ (py) ₄ ¹⁺ . 2. The method of claim 1, wherein the step of reacting comprises reacting the hybrid DNA with the transition metal complex under conditions sufficiently effective for selective oxidation of the preselected base. 2. The method of claim 1, further comprising propagating the hybrid DNA before the contacting step. 10. The method according to claim 9, wherein the step of propagating the DNA sample is carried out by a polymerase chain reaction, a chain replacement proliferation, a ligase chain reaction, or a proliferation based on a nucleic acid sequence. 3. The method of claim 2, wherein measuring the reaction rate of the oxidation-reduction reaction comprises measuring a cyclic volt-ampere of the reaction. 3. The method of claim 2, wherein said comparing step comprises comparing the cyclic voltage current of the reaction of the transition metal complex with the hybrid DNA sample and the known cyclic voltage current of the reaction of the transition metal complex with single-chain DNA Way. 2. The method of claim 1, wherein the oligonucleotide probe is immobilized on a solid surface. 14. The method of claim 13, wherein the transition metal complex is fixed on the solid surface. 2. The method of claim 1, further comprising: (e) identifying a base pairing with the preselected base. 2. The method of claim 1, further comprising: (e) identifying a base paired with a base adjacent to the preselected base. 17. The method of claim 15 or 16, wherein identifying the base comprises the steps of (i) measuring the detected oxidation-kinetics rate, and (ii) determining the amount of adenine, cytosine, guanine, or thymine base (Iii) measuring whether the measured reactivity is the same as the known oxidation-reduction reaction rate, and (ii) comparing the measured oxidation- ≪ / RTI > (a) contacting a DNA sample with an oligonucleotide probe to form hybrid DNA; (b) reacting the hybrid DNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having at least one preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the detected oxidation-reaction rate; (e) comparing the measured reaction rate with the rate of oxidation-reduction reaction of the transition metal complex and single-chain DNA; And (f) measuring whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the transition metal complex and the single-chain DNA. 19. The method of claim 18, wherein the preselected base is guanine. 19. The method of claim 18, wherein the preselected base is adenine. 19. The method of claim 18, wherein the transition metal complex is ^{_{Ru (bpy) 3 2+, Ru}} (Me 2 -bpy) 3 2+, Ru (Me 2 -phen) 3 2+, Fe (bpy) 3 2+, Fe (5-Cl-phen) ₃ ²⁺ , Os (5-Cl-phen) ₃ ²⁺ , and ReO ₂ (py) ₄ ¹⁺ . 19. The method of claim 18, wherein the step of reacting comprises reacting the hybrid DNA with the transition metal complex under conditions sufficiently effective for selective oxidation of the preselected base. 19. The method of claim 18, further comprising propagating the hybrid DNA prior to the contacting step. 24. The method according to claim 23, wherein the step of propagating the DNA sample is performed by a polymerase chain reaction, a chain substitution proliferation, a ligase chain reaction, or a proliferation based on a nucleic acid sequence. 19. The method of claim 18, wherein measuring the reaction rate of the oxidation-reduction reaction comprises measuring a cyclic volt-ampere of the reaction. 19. The method of claim 18, wherein the step of comparing comprises comparing the cyclic voltage current of the reaction of the transition metal complex with the hybrid DNA sample and the known cyclic voltage current of the reaction of the transition metal complex with the single- Way. 19. The method of claim 18, wherein the oligonucleotide probe is immobilized on a solid surface. 28. The method of claim 27, wherein the transition metal complex is fixed on the solid surface. 19. The method of claim 18, further comprising (g) identifying a base that pairs with the preselected base. 30. The method of claim 29, wherein step (g) of identifying the base comprises the steps of: (i) determining four different known reaction rates of the DNA having the adenine, cytosine, guanine, or thymine base bound to the preselected base and the transition metal complex, And (ii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate. (a) a plurality of DNA sample containers; (b) sample manipulating means for transferring the plurality of DNA sample containers; (c) oligonucleotide probe transfer means for transferring the oligonucleotide probe to each of the DNA sample containers; (d) transition metal complex transfer means for transferring the transition metal complex to each of the DNA sample containers; And (e) an oxidation-reduction reaction detector for detecting oxidation-reduction reaction. 32. The apparatus of claim 31, further comprising a measurement means for measuring the oxidation-reduction reaction rate to detect the detected oxidation-reduction reaction rate. 32. The apparatus of claim 31, wherein the oxidation-reduction reaction detector is an electrode. 32. The apparatus of claim 31, wherein the oligonucleotide probe transfer means comprises a solid surface having an oligonucleotide probe that does not move thereon. (a) a DNA sample container; (b) an oligonucleotide probe transfer means for transferring the oligonucleotide probe to the DNA sample container; (c) transition metal complex transporting means for transporting the transition metal complex to the DNA sample vessel; And (d) an oxidation-reduction reaction detector for detecting an oxidation-reduction reaction. 36. The apparatus of claim 35, further comprising a redox reaction measuring the detected oxidation-reduction reaction rate. 36. The apparatus of claim 35, wherein the oxidation-reduction reaction detector is an electrode. 36. The apparatus of claim 35, wherein the oligonucleotide probe transfer means comprises a solid surface having a plurality of different oligonucleotide probes that do not migrate thereon. 39. The apparatus of claim 38, wherein the transition metal complex transport means comprises a solid surface having a plurality of oligonucleotide probes and a transition metal complex that do not move thereon. (a) contacting a DNA sample with an oligonucleotide probe comprising a preselected base having a unique oxidation potential to form hybrid DNA; (b) reacting the hybrid DNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having a preselected number of preselected bases in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the reaction rate of the detected oxidation-reduction reaction; And (e) identifying a base paired with said preselected base. 42. The method of claim 40, wherein said identifying step comprises the steps of: (i) determining four different known reaction rates of each of the transition metal complexes with DNA having an adenine, cytosine, guanine, or thymine base bound to the preselected base, And (ii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate. 41. The method of claim 40, wherein the oligonucleotide probe comprises a second preselected base having an intrinsic oxidation potential different from the oxidation potential of the preselected base. 43. The method of claim 42, wherein the detecting step further comprises detecting an oxidation-reduction reaction between the transition metal complex and the second preselected base, Wherein the measuring step further comprises measuring the rate of the oxidation-reduction reaction between the transition metal complex and the second preselected base; Wherein the identifying step further comprises identifying a base pairing with the second preselected base. 41. The method of claim 40, further comprising repeating steps (a) to (e) with a sufficient number of oligonucleotide probes having the preselected base at a different site that identifies each base in the DNA sample. (a) contacting an RNA sample with an oligonucleotide probe to form a hybrid RNA; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having at least one preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the presence of the hybrid RNA from the oxidation-reduction reaction detected in the preselected base. 48. The method of claim 45, wherein said measuring step comprises the steps of: (i) measuring the rate of the reaction of the detected oxidation reaction; (ii) determining the ratio of RNA having an adenine, cytosine, guanine, or thymine base bound to the pre- And (iii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate, characterized by comprising the steps of: How to. 46. The method of claim 45, wherein the RNA sample is a single-stranded RNA sample and the hybrid RNA is a double-stranded. 46. The method of claim 45, wherein the oligonucleotide probe comprises 4 to 100 bases. 46. The method of claim 45, wherein said preselected base is guanine. 46. The method of claim 45, wherein the preselected base is adenine. The method of claim 45 wherein the transition metal complex is ^{_{Ru (bpy) 3 2+, Ru}} (Me 2 -bpy) 3 2+, Ru (Me 2 -phen) 3 2+, Fe (bpy) 3 2+, Fe (5-Cl-phen) ₃ ²⁺ , Os (5-Cl-phen) ₃ ²⁺ , and ReO ₂ (py) ₄ ¹⁺ . 46. The method of claim 45, wherein said step of reacting comprises reacting said hybridization RNA with said transition metal complex under conditions sufficiently effective for selective oxidation of said preselected base. 46. The method of claim 45, further comprising propagating the hybrid RNA prior to the contacting step. 49. The method according to claim 48, wherein the step of propagating the RNA sample is performed by a reverse-transcription enzyme-polymerase chain reaction. 47. The method of claim 46, wherein measuring the reaction rate of the oxidation-reduction reaction comprises measuring a cyclic volt-ampere of the reaction. 47. The method of claim 46, wherein said comparing step comprises comparing the cyclic voltage current of the reaction of the transition metal complex with the hybrid RNA sample and the known cyclic voltammetric of the reaction of the transition metal complex with the single-chain RNA Way. 46. The method of claim 45, wherein the oligonucleotide probe is immobilized on a solid surface. 58. The method of claim 57, wherein the transition metal complex is immobilized on the solid surface. 46. The method of claim 45, further comprising: (e) identifying a base paired with the preselected base. 46. The method of claim 45, further comprising: (e) identifying a base paired with a base adjacent to the preselected base. 46. The method of claim 45, wherein said identifying step comprises the steps of: (i) measuring the rate of said detected oxidation reaction to determine (ii) the RNA having an adenine, cytosine, guanine, or thymine base bound to said pre- And (iii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate, characterized by comprising the steps of: How to. (a) contacting an RNA sample with an oligonucleotide probe to form a hybrid RNA; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing a preselected base in the nucleotide having at least one preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; (d) measuring the detected oxidation-reaction rate; (e) comparing the measured reaction rate with the rate of oxidation-reduction reaction of the transition metal complex and single-chain RNA; (f) measuring whether the measured reaction rate is equal to the oxidation-reduction reaction rate of the transition metal complex and the single-chain RNA. 63. The method of claim 62, wherein said preselected base is guanine. 63. The method of claim 62, wherein the preselected base is adenine. The method of claim 62 wherein the transition metal complex is ^{_{Ru (bpy) 3 2+, Ru}} (Me 2 -bpy) 3 2+, Ru (Me 2 -phen) 3 2+, Fe (bpy) 3 2+, Fe (5-Cl-phen) ₃ ²⁺ , Os (5-Cl-phen) ₃ ²⁺ , and ReO ₂ (py) ₄ ¹⁺ . 63. The method of claim 62, wherein said step of reacting comprises reacting said hybridization RNA with said transition metal complex under conditions sufficiently effective for selective oxidation of said preselected base. 63. The method of claim 62, further comprising propagating the hybrid RNA prior to the contacting step. 68. The method of claim 67, wherein the step of propagating the RNA sample is performed by a polymerase chain reaction, a chain replacement proliferation, a ligase chain reaction, or a proliferation based on a nucleic acid sequence. 63. The method of claim 62, wherein measuring the reaction rate of the oxidation-reduction reaction comprises measuring a cyclic volt-ampere of the reaction. 63. The method of claim 62, wherein said comparing step comprises comparing the cyclic voltage current of the reaction of the transition metal complex with the hybrid RNA sample and the known cyclic voltage current of the reaction of the transition metal complex with the single- Way. 63. The method of claim 62, wherein the oligonucleotide probe is immobilized on a solid surface. 74. The method of claim 71, wherein the transition metal complex is immobilized on the solid surface. 63. The method of claim 62, further comprising: (g) identifying the base paired with the preselected base. 72. The method of claim 73, wherein step (g) of identifying the base comprises the steps of: (i) determining at least four known reaction rates of each of the RNAs having an adenine, cytosine, guanine, or thymine base bound to the preselected base, And (ii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate. (a) contacting an RNA sample with an oligonucleotide probe comprising a preselected base having a unique oxidation potential to form a hybrid RNA; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing in a redox reaction a preselected base in the nucleotide having a preselected number of preselected bases; (c) detecting the oxidation-reduction reaction; (d) measuring the reaction rate of the detected oxidation-reduction reaction; And (e) identifying a base paired with said preselected base. 76. The method of claim 75, wherein the step of identifying comprises: (i) determining four different known reaction rates of each of the RNAs having the adenine, cytosine, guanine, or thymine base bound to the preselected base and the transition metal complex, And (ii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate. 76. The method of claim 75, wherein the oligonucleotide probe comprises a second preselected base having an intrinsic oxidation potential different from the oxidation potential of the preselected base. 78. The method of claim 77, wherein said detecting step further comprises detecting an oxidation-reduction reaction between the transition metal complex and the second preselected base, Wherein the measuring step further comprises measuring the rate of the oxidation-reduction reaction between the transition metal complex and the second preselected base; Wherein the identifying step further comprises identifying a base pairing with the second preselected base. 76. The method of claim 75, further comprising repeating steps (a) to (e) with a sufficient number of oligonucleotide probes having the preselected base at a different site to identify each base in the RNA sample. (a) reacting the nucleic acid with a transition metal complex capable of oxidizing a preselected base in an oxidation-reduction reaction; (b) detecting the oxidation-reduction reaction; And (c) measuring the presence or absence of said nucleic acid from the oxidation-reduction reaction detected in said preselected base. 81. The method of claim 80, wherein the step of reacting is carried out by contacting the nucleic acid with a complementary nucleic acid to form a hybrid nucleic acid. 83. The method of claim 81, wherein the measuring step comprises the steps of (i) measuring the detected oxidation-kinetics rate, and (ii) comparing the measured kinetics with the rate of oxidation-reduction kinetics of the transition metal complex and the single- , And (iii) measuring whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the transition metal complex and the single-chain nucleic acid. 83. The method of claim 82, wherein measuring the reaction rate of the oxidation-reduction reaction comprises measuring a cyclic volt-ampere of the reaction. 83. The method of claim 82, wherein said comparing step comprises comparing the cyclic voltage current of the reaction of the transition metal complex with the hybrid nucleic acid sample and the known cyclic voltage current of the reaction of the transition metal complex with the single-chain nucleic acid Way. 83. The method of claim 81, wherein after said measuring step, identifying a base pairing with a previously selected base is performed. 83. The method of claim 81, wherein after said measuring step, identifying a base pairing with a base adjacent to a preselected base is performed. 85. The method of claim 85 or 86, wherein the identifying step comprises the steps of (i) measuring the rate of the reaction of the detected oxidation reaction, (ii) contacting the nucleic acid having an adenine, cytosine, guanine, or thymine base bound to the pre- Comparing the four different known reaction rates of the transition metal complex with the measured oxidation-reaction rate, and (iii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate Lt; / RTI > 79. The method of claim 80, wherein the nucleic acid comprises 4 to 100 bases. 79. The method of claim 80, wherein the preselected base is selected from the group consisting of guanine and adenine. The method of claim 80, wherein the transition metal complex is ^{_{Ru (bpy) 3 2+, Ru}} (Me 2 -bpy) 3 2+, Ru (Me 2 -phen) 3 2+, Fe (bpy) 3 2+, Fe (5-Cl-phen) ₃ ²⁺ , Os (5-Cl-phen) ₃ ²⁺ , and ReO ₂ (py) ₄ ¹⁺ . 83. The method of claim 80, wherein the nucleic acid is DNA. 79. The method of claim 80, wherein the nucleic acid is RNA. 83. The method of claim 80, wherein said step of reacting comprises reacting said nucleic acid with said transition metal complex under conditions sufficiently effective for selective oxidation of said preselected base. 79. The method of claim 80, further comprising propagating the nucleic acid prior to the contacting step. 95. The method of claim 94, wherein the step of propagating the nucleic acid is performed by a polymerase chain reaction, a chain substitution proliferation, a ligase chain reaction, or a proliferation based on a nucleic acid sequence. 83. The method of claim 80, wherein the nucleic acid is immobilized on a solid surface. 96. The method of claim 96, wherein the transition metal complex is immobilized on the solid surface. (a) contacting the test sample with an oligonucleotide probe that specifically binds to a target nucleic acid further comprising at least 10 bases selected from the oligonucleotide probes, to form a hybrid nucleic acid; (b) contacting the hybridized nucleic acid with a transition metal complex which oxidizes the preselected base in an oxidation-reduction reaction; (c) detecting the oxidation-reduction reaction; And (d) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base, in the test sample. 98. The method of claim 98, further comprising separating the test sample from the hybrid nucleic acid prior to the detecting step. 98. The method of claim 98, wherein the target nucleic acid comprises more than 10 of the pre-selected bases than the oligonucleotide probe. 98. The method of claim 98, wherein the oligonucleotide probe is free from the preselected base. 97. The method of claim 98, wherein the target nucleic acid is longer than the oligonucleotide probe and wherein at least one of the pre-selected bases is not hybridized to the oligonucleotide probe in the hybrid nucleic acid. 97. The method of claim 98, wherein the measuring step is a quantitative measuring step. 98. The method of claim 98, wherein the measuring step comprises the steps of: (i) measuring the rate of the reaction of the detected oxidation reaction to determine the rate of oxidation-reduction reaction of the single-chain target nucleic acid and the transition metal complex, And (iii) measuring whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the single-chain target nucleic acid and the transition metal complex. 105. The method of claim 104, wherein measuring the reaction rate of the oxidation-reduction reaction comprises measuring the cyclic volt-ampere of the reaction. 105. The method of claim 104, wherein said comparing step comprises comparing the cyclic voltage current of the reaction of the transition metal complex with the hybrid target nucleic acid and the known cyclic voltammogram of the single-chain target nucleic acid with the transition metal complex . 98. The method of claim 98, wherein the nucleic acid comprises 4 to 100 bases. 98. The method of claim 98, wherein the preselected base is selected from the group consisting of guanine and adenine. The method of claim 98, wherein the transition metal complex is ^{_{Ru (bpy) 3 2+, Ru}} (Me 2 -bpy) 3 2+, Ru (Me 2 -phen) 3 2+, Fe (bpy) 3 2+, Fe (5-Cl-phen) ₃ ²⁺ , Os (5-Cl-phen) ₃ ²⁺ , and ReO ₂ (py) ₄ ¹⁺ . 98. The method of claim 98, wherein the target nucleic acid is DNA. 98. The method of claim 98, wherein the target nucleic acid is RNA. 98. The method of claim 98, wherein the step of reacting comprises reacting the target nucleic acid with the transition metal complex under conditions sufficiently effective for selective oxidation of the preselected base. 98. The method of claim 98, further comprising propagating the target nucleic acid prior to the contacting step. 113. The method according to claim 113, wherein the step of propagating the nucleic acid is performed by a polymerase chain reaction, a chain replacement proliferation, a ligase chain reaction, or a proliferation based on a nucleic acid sequence. 98. The method of claim 98, wherein the oligonucleotide probe is immobilized on a solid surface. 116. The method of claim 115, wherein the transition metal complex is immobilized on the solid surface. (a) contacting the test sample with a complementary nucleic acid that specifically binds the target nucleic acid to form a hybrid nucleic acid; (b) contacting the hybrid nucleic acid with a transition metal complex which oxidizes the preselected base in an oxidation-reduction reaction; (c) detecting the presence of an oxidation-reduction reaction associated with the hybrid nucleic acid; And (d) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base, in a test sample to detect a target nucleic acid containing at least one preselected base As a result, Wherein the preselected base in the target nucleic acid is guanine; Wherein the target nucleic acid comprises cytosine; Said oligonucleotide probe comprising a corresponding base which binds to a cytosine in said hybrid nucleic acid; Wherein the corresponding base is selected from the group consisting of inosine and 7-deaza-guanine. 117. The method of claim 117, wherein the step of reacting comprises reacting the transition metal complex with the nucleic acid under conditions effective for selective oxidation of the preselected base, without oxidation of the corresponding base. 117. The method of claim 117, wherein said measuring step comprises the steps of: (i) measuring the rate of said detected oxidation reaction to determine (ii) the rate of oxidation-reduction reaction of the single- And (iii) measuring whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the single-chain target nucleic acid and the transition metal complex. 111. The method of claim 105, wherein measuring the reaction rate of the oxidation-reduction reaction comprises measuring a cyclic volt-ampere of the reaction. 119. The method of claim 119, wherein said comparing step comprises comparing the cyclic voltage current of the reaction of the transition metal complex with the hybrid target nucleic acid and the known cyclic voltage current of the single-chain target nucleic acid with the transition metal complex . 117. The method of claim 117, wherein after said measuring step, identifying a base pairing with a pre-selected base is performed. 117. The method of claim 117, wherein after said measuring step, identifying a base pairing with a base adjacent to a preselected base is performed. 122. The method of claim 122 or claim 123, wherein the identifying step comprises the steps of (i) measuring the rate of the reaction of the detected oxidation reaction, (ii) contacting the nucleic acid having an adenine, cytosine, guanine, or thymine base bound to the pre- Comparing the four different known reaction rates of the transition metal complex with the measured oxidation-reaction rate, and (iii) measuring whether the measured reaction rate is the same as the known oxidation-reduction reaction rate Lt; / RTI > 117. The method of claim 117, wherein the nucleic acid comprises 4 to 100 bases. The method of claim 117, wherein the transition metal complex is ^{_{Ru (bpy) 3 2+, Ru}} (Me 2 -bpy) 3 2+, Ru (Me 2 -phen) 3 2+, Fe (bpy) 3 2+, Fe (5-Cl-phen) ₃ ²⁺ , Os (5-Cl-phen) ₃ ²⁺ , and ReO ₂ (py) ₄ ¹⁺ . 118. The method of claim 117, wherein the target nucleic acid is DNA. 118. The method of claim 117, wherein the target nucleic acid is RNA. 117. The method of claim 117, further comprising propagating the target nucleic acid prior to the contacting step. 129. The method according to claim 129, wherein the step of propagating the nucleic acid is performed by a polymerase chain reaction, a chain substitution proliferation, a ligase chain reaction, or a proliferation based on a nucleic acid sequence. 117. The method of claim 117, wherein the complementary nucleic acid is immobilized on a solid surface. 132. The method of claim 131, wherein the transition metal complex is immobilized on the solid surface. (a) contacting a test sample with an oligonucleotide probe that specifically binds to the target nucleic acid and has an end blocked by a terminal transferase to form a hybrid nucleic acid; (b) contacting the hybrid nucleic acid with a solution containing a base selected in advance in the presence of a terminal transferase to produce an extension product consisting of a preselected base; (c) contacting the oligonucleotide probe with a transition metal complex that oxidizes the preselected base in a redox reaction; (d) detecting the presence of the target nucleic acid in an oxidation-reduction reaction; And (e) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. 133. The method of claim 133, further comprising separating the test sample from the hybrid nucleic acid prior to the detecting step. (a) providing a capture probe that specifically binds a target nucleic acid; (b) contacting the test sample with the capture probe to form a hybrid nucleic acid; (c) contacting the oligonucleotide signal probe, which specifically binds to the target nucleic acid and contains one or more preselected bases, to the hybrid nucleic acid to generate a hybrid nucleic acid sandwich; (d) contacting the hybrid nucleic acid sandwich with a transition metal complex that oxidizes the preselected base in an oxidation-reduction reaction; (e) detecting the presence of an oxidation-reduction reaction associated with the hybrid nucleic acid sandwich; And (f) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. 136. The method of claim 135, further comprising separating the test sample from the hybrid nucleic acid prior to the detecting step. 136. The method of claim 136, wherein said separating step is performed between step (b) and step (c) or between step (c) and step (d). A microelectronic substrate having first and second opposing surfaces; A conductive electrode on the first side; And A microelectronic device for electrochemically detecting a nucleic acid species comprising an oligonucleotide capture probe immobilized on a first surface adjacent to the conductive electrode. 143. The apparatus of claim 138, wherein the apparatus has a plurality of conductive electrodes on a first side and a plurality of oligonucleotide capture probes located adjacent to the different conductive electrodes and secured on the first side. . 136. The apparatus of claim 138, further comprising a contact electrically connected to the conductive electrode. 136. The apparatus of claim 138, wherein the substrate is silicon. 128. The apparatus of claim 123, wherein the oligonucleotide capture probe is 4 to 100 nucleic acid lengths. Oligonucleotide probes useful for electrochemical detection of preselected bases in the presence of background guanine signal, comprising at least one purine base of formula (I): 143. The apparatus of claim 143, wherein the oligonucleotide capture probe is 100 nucleotides or less in length. (a) providing a nucleic acid comprising at least one base of the formula: (b) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in a redox reaction; (c) detecting the oxidation-reduction reaction; And (d) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. 145. The method of claim 145, wherein the step of reacting comprises contacting the nucleic acid with a complementary nucleic acid to form a hybrid nucleic acid. 145. The method of claim 146, wherein the complementary nucleic acid comprises at least one guanine substituent. (a) a conductive substrate having a work surface; And (b) a non-conducting polymer layer connected to the working surface, the non-conducting polymer layer being coplanar with the transition metal complex, An electrode useful for electrochemical detection of a preselected base in a nucleic acid by reacting the nucleic acid with a transition metal complex capable of oxidizing a preselected base in an oxidation-reduction reaction. 143. The method of claim 148, further comprising a nucleic acid bound to the polymeric layer and comprising at least one of the pre-selected bases. (a) contacting a sample comprising nucleic acid with (i) a conductive substrate having a working surface; And (ii) a non-conducting polymer layer connected to the working surface and bonded to a nucleic acid; (b) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in a redox reaction and wherein the polymer layer is pore-free therewith; (c) detecting the oxidation-reduction reaction by measuring a current flowing through the electrode; And (d) measuring the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. 153. The method of claim 150, wherein the step of contacting comprises contacting the nucleic acid with a complementary nucleic acid to form a hybrid nucleic acid.