JP2004117371A - Electrochemical detection of nucleic acid hybridization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method for nucleic acids containing at least one specific base (for example, adenine, guanine, 6-mercaptoguanine, 8-oxo-quanine, 8-oxo-adenine). <P>SOLUTION: The method comprises (a) a stage in which a nucleic acid reacts with transition metal complexes enabling to oxidize a specific base under an oxidation-reduction reaction; (b) a stage in which the oxidation-reduction reaction is detected; (c) a stage in which presence or absence of the nucleic acids is determined from the oxidation-reduction reaction detected with the specific base. A method is provided for detecting nucleic acids (for example, DNA, RNA) containing at least one specific base (for example, adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, 8-oxo-adenine). The method is used in versatile applications including DNA sequencing, diagnostic analysis and quantitative analysis. <P>COPYRIGHT: (C)2004,JPO

Description

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

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

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

U.S. Pat. No. 4,840,893 to Hill et al. Describes an electrochemical assay for nucleic acids in which the competitive binding behavior between the ligand and the anti-ligand is sequentially detected electrochemically. .

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

Generally, the present invention provides a method for detecting a nucleic acid containing at least one predetermined base (eg, adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine). The method comprises the steps of: (a) reacting a nucleic acid with a transition metal complex capable of oxidizing a predetermined base in a redox reaction; (b) detecting the redox reaction; and (c) detecting a predetermined base. Determining the presence or absence of the nucleic acid from the oxidation-reduction reaction detected in step (1). Depending on the particular embodiment of the method and the particular purpose desired, the invention further comprises the step of contacting the nucleic acid with a supplemental nucleic acid to form a hybridizing nucleic acid.

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

According to a second aspect, the present invention provides another method for detecting DNA hybridization. The method includes: (a) contacting a DNA sample with an oligonucleotide probe to form hybridized DNA; and (b) the oligonucleotide probe has at least one predetermined base and oxidizes the hybridized DNA. Reacting with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe in a reduction reaction, (c) detecting a redox reaction, and (d) reacting the detected redox reaction Measuring the rate; (e) comparing the measured reaction rate with the rate of the redox reaction of the transition metal complex with the single-stranded DNA; and (f) determining the measured reaction rate of the single-stranded DNA. Determining whether the rate of the oxidation-reduction reaction of the transition metal complex is substantially the same as that of the transition metal complex.

According to a third aspect, the present invention provides an apparatus for detecting DNA hybridization. The apparatus comprises (a) a plurality of DNA sample containers, (b) sample handling means for carrying the plurality of DNA sample containers, and (c) an oligo for delivering oligonucleotide probes to each of the DNA sample containers. A nucleotide probe delivery means, (d) a transition metal complex delivery means for delivering a transition metal complex to each of the plurality of DNA sample containers, and (e) an oxidation-reduction reaction detector for detecting an oxidation-reduction reaction. including.

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

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

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

The term "nucleic acid" as used herein refers to any nucleic acid, including both DNA and RNA. The nucleic acids of the invention are typically polynucleic acids, macromolecules of individual nucleotides covalently linked by 3 ', 5' phosphodiester bonds.

"Complementary nucleic acid" as used herein refers to any nucleic acid, including an oligonucleotide probe, that specifically binds to another nucleic acid to make a hybridizing nucleic acid.

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

The terms "hybridized DNA" and "hybridized nucleic acid" refer to single-stranded DNA or triple-helix DNA or nucleic acid that is hybridized to form double-stranded DNA or nucleic acid. A double-stranded DNA or nucleic acid that is hybridized to form.

Although the methods and devices of the present invention are sometimes described herein with respect to DNA, this is for clarity and it is understood that the present methods and devices can be applied to other nucleic acids, such as RNA. It should be understood.

A. Nucleic Acid Amplification Method Since the process of the present invention involves contacting a DNA sample with an oligonucleotide probe to produce hybridized DNA, certain applications for amplifying DNA prior to contact with the probe are desirable. Amplification of the selected or desired nucleic acid sequence can be performed by any suitable means. Generally, Kwoh and T.W. Kwoh's Am. Biotechnol. Lab. See Vol. 8, pp. 14-25 (1990). Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction (including reverse transcriptase chain reaction on RNA), ligase chain reaction, strand displacement amplification, transcriptase amplification (D, Kwoh et al., Proc. Natl. USA 86, 1173-1177 (1989), self-supported sequence replication (or "3SR") (J. Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874-178). (1990)), the Qβ replicase system (see P. Lizardi et al., Biotechnology 6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or “NASBA”) (R. Lewis Genetic). Engineering News 12 ( 9), p. 1 (1992), repair chain reaction (or "RCR") (see above, R. Lewis), and boomerang DNA amplification (or "BDA") (see above, R. Lewis) The base incorporated in the applied product is a natural or modified base (before amplification or modified to sound) and is selected to optimize the subsequent electrochemical detection step. .

The polymerase chain reaction (PCR) is also performed with known techniques. See, for example, U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (for examples of all U.S. Pat. The disclosure is to be incorporated as an example.) In general, PCR involves initially binding one strand to each strand of a specific sequence to be detected under hybridization conditions, such that an extension product of each primer that is complementary to each strand of nucleic acid is synthesized. Treating one oligonucleotide primer and a nucleic acid sample (eg, in the presence of a thermostable DNA polymerase), wherein the primers bind to each other when the extension product synthesized from each primer is separated from its complement Is sufficiently complementary to each strand of the specific sequence to hybridize so that it can serve as a template for the synthesis of the extension product of Treating the samples under denaturing conditions to separate the primer extension products from their templates, if present. These steps are repeated periodically until the desired degree of amplification is obtained. Detection of the amplified sequence is accomplished by adding an oligonucleotide probe (eg, an oligonucleotide probe of the invention) that is capable of hybridizing to the reaction product and carrying a detectable label to the reaction product, and then This is done by detecting the label according to the technique. If the nucleic acid to be amplified is RNA, amplification is performed by initial conversion to DNA by reverse transcription according to known techniques.

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

Perigase chain reaction (LCR) is also performed according to known techniques. For example, R. See Weiss Science, Vol. 254, p. 1292 (1991). Generally, one pair binds to one strand of the sequence to be detected and the other pair binds to the other strand of the sequence to be detected: performed using two pairs of oligonucleotide probes. Each pair together completely overlaps the corresponding strand. The reaction first denatures (e.g., separates) the strands of the sequence to be detected, and then ligates the strands in the presence of a thermostable ligase so that each pair of oligonucleotide probes is bound together. React with the nucleotide probe and separate the reaction product therefrom, after which the process is repeated periodically until the sequence is amplified to the desired degree. Detection is then performed in a similar manner as described above for PCR.

B. Oligonucleotide Probes As described above, the production method of the present invention is useful for detecting DNA hybridization. The first step of the manufacturing method involves contacting a DNA sample with an oligonucleotide probe to form hybridized DNA. Oligonucleotide probes useful in the methods of the invention comprise between about 4 or 6 bases and about 80 or 100 bases or more, more preferably between about 8 and about 15 bases. Probe. Oligonucleotide probes can be prepared having any of a wide variety of base sequences according to techniques well known in the art. Suitable bases for the preparation of oligonucleotide probes include naturally occurring nucleotide bases such as adenosine, cytosine, guanine, uracil, and thymine; and 8-oxo-guanine, 6-mercaptoguanine, 4-acetylcytidine, 5- ( (Carboxyhydroxyethyl) uridine, 2′-O-methylcytidine, 5-carboxymethylamino-methyl-2-thiolidine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2′-O-methylpseudouridine, β, D -Galactosylkeosin, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2- Tilguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, β, D-mannosylkeosin, 5- Methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl) carbamoyl) threonine, N-((9 -Β-D-ribofuranosylpurin-6-yl) N-methyl-carbamoyl) threonine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, wybutoxosine, pseudouridine, keosin , 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 2-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl) carbamoyl) threonine, 2 ′ Non-naturally occurring or "synthetic" nucleotide bases such as -O-methyl-5-methyluridine, 2'-O-methyluridine, wibutosine, and 3- (3-amino-3-carboxypropyl) uridine Is selected from Oligonucleotide backbones are used including DNA, RNA (although RNA is less preferred than DNA), improved sugars such as carbocycles, and sugars containing 2 'substitutions such as fluoro and methoxy. Oligonucleotides have at least one or all of the internucleotide linkage phosphate residues in which methylphosphonate, methylphosphonothioate, phosphoromorpholidate, phosphoropiperazidate, phosphoramidate, etc. Oligonucleotides which are improved phosphates wherein the internucleotide linkage phosphate residues are improved as described). Oligonucleotides are P.I. "Peptide nucleic acid" as described in Nielsen et al., Science 254, pp. 1497-1500 (1991). The oligonucleotide probe need only at least partially retain a sequence capable of binding at least a known portion of the sequence of the DNA sample. It can be used in some applications where a DNA sample is combined with a number of oligonucleotide probes having different base sequences (eg, when two or more nucleic acids of interest are present in a sample, or when a single nucleic acid of interest is "sandwiched"). (Where hybridized to two or more probes).

C. Hybridization Methodology A DNA (or nucleic acid) sample is contacted with an oligonucleotide probe in any suitable manner known to those skilled in the art. For example, a DNA sample is dissolved in a solution and contacted with the oligonucleotide probe by dissolving the oligonucleotide probe in a solution containing the DNA sample under conditions that permit hybridization. Suitable conditions are known to those skilled in the art (see, for example, U.S. Pat. No. 4,358,535 to Falkow et al. And other U.S. Patents which describe the same) and include high salt concentration conditions. . Alternatively, a DNA sample is dissolved in a solution with the oligonucleotide probe immobilized on a solid support, and the DNA sample is immersed in a solution containing the DNA sample with the oligonucleotide probe immobilized thereon. Thereby contacting the oligonucleotide probe.

D. Oxidizing Agent and Redox Reaction As the hybridization step proceeds to the oxidation step, after hybridization, the hybridized DNA (or nucleic acid) is suitably redox-oxidized to a predetermined base in the oligonucleotide probe. Reacts with oxidants. The predetermined base can be a naturally occurring or synthetic nucleotide base in the oligonucleotide probe that undergoes oxidation in reaction with the selected oxidizing agent. Certain bases exhibit a unique rate of oxidation when a given base is paired compared to when it is unpaired. Certain bases exhibit unique oxidation rates when paired with each of the four naturally occurring bases. In general, 5'-mononucleotides (e.g., 5'-deoxyribonucleotide or 5'-ribonucleotide) bases show a detectable ¹⁰ 4 ^{M -1 s} -1 or more rate constants using a catalytic reaction. Examples of suitable predetermined bases include, but are not limited to, guanine, adenosine, 8-oxo-guanine, and 8-oxo-adenosine, 8-bromo-guanine, guanosine, xanthosine, viosin, pseudouridine, 6-mercaptoguanine, 8-mercaptoguanine, 2-thioxanthine, 6-thioxanthine, 6-mercaptopurine, 2-amino-6-carboxymethyl-mercaptopurine, 2-mercaptopurine, 6-methoxypurine, 2-acetylamino-6-hydroxy Purine, 6-methylthio-2-hydroxypurine, 2-dimethylamino-6-hydroxypurine, 2-hydroxypurine, 2-aminopurine, 6-amino-2-dimethylallyl-purine, 2-thioadenine, 8-hydroxyadenine , Containing 8-methoxyadenine Typically, the predetermined base is selected from the group consisting of guanine, adenosine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine, where guanine is a currently preferred naturally occurring predetermined base. Yes, 6-mercaptoguanine is the currently preferred synthetic base of choice.

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

The oxidizing agent is reacted with the hybridized DNA according to a suitable technique to achieve a redox reaction of the oxidizing agent with a predetermined base. All that is required is that the oxidizing agent be reacted with the hybridized DNA under conditions effective to achieve selective oxidation of the given base. For example, the transition metal is dissolved by dissolving the oxidizing agent in a solution containing the dissolved hybridized DNA under conditions sufficient to cause a redox reaction between the oxidizing agent and a given base. Is reacted with the hybridized DNA. In another embodiment, in which DNA is immobilized on a solid support, an oxidizing agent is immobilized on the same solid support, and the solid support is used to cause a redox reaction between the oxidizing agent and a predetermined base. The oxidizing agent is reacted with the hybridized DNA by immersion in the solution under sufficient conditions. The solvent in which the oxidation-reduction reaction occurs is a solvent suitable for dissolving DNA, and preferably comprises water. Conditions suitable for causing a redox reaction are known to those skilled in the art.

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

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

検 出 Detection of the electrical signal associated with the redox reaction allows for the determination of the presence or absence of the hybridized DNA. Determining the presence or absence of the hybridizing DNA typically includes (i) measuring the kinetics of the redox reaction, and (ii) measuring the kinetics of the redox reaction with the transition metal with the single-stranded DNA. Comparing the redox reaction rate of the complex with the redox reaction rate of the complex; and (iii) determining whether the measured reaction rate is substantially the same as the redox reaction rate of the transition metal complex with the single-stranded DNA. Including. The step of measuring the reaction rate is performed by any suitable means. For example, relative reaction rates are determined by comparing currents as a function of sweep rate, probe concentration, target concentration, media, buffers, temperature, and / or electrochemical methods.

The oxidation-reduction rate is measured according to an appropriate means known to those skilled in the art. Typically, the redox reaction rate is measured by measuring an electrical signal coupled to the occurrence of the redox reaction. For example, the electrical signal coupled to the redox reaction is measured by providing a suitable device in electrical communication with the sensing electrode. Suitable devices can measure the electrical signal generated to provide a measure of the redox kinetics of the reaction between the hybridizing DNA and the oxidizing agent. The electrical signal is specific to any of the electrochemical methods, including cyclic voltammetry, conventional pulse voltammetry, chronoamperometry, and square wave voltammetry, and cyclic voltammetry is a recently preferred form .

The measured kinetics are then compared to the known redox kinetics of the transition metal complex with single-stranded DNA. As already discussed in detail, in either hybridized or single-stranded DNA, the tunneling distance between the oxidizing agent and the predetermined base depends on the redox reaction of the reaction between the oxidizing agent and the predetermined base. Affects speed. Therefore, the hybridized DNA exhibits a different redox reaction rate than the single-stranded DNA. The presence or absence of the hybridized DNA at a given base is based on whether the measured redox reaction rate is the same as the redox reaction rate between the oxidizing agent and the given base in the single-stranded DNA. Can be determined. Further, the tunneling distance between the oxidizing agent and a given base will vary according to the binding distance between a given base and its partner such that each possible base pairing is distinguished from the others. The binding distance between a given base and its base pair depends on the base paired with the given base. For example, the redox rate for the oxidation of guanine paired with adenosine is different from the redox rate for the oxidation of guanine paired with cytosine, and then the redox rate for the oxidation of guanine paired with cytosine is guanine. The redox rate for the oxidation of guanine paired with guanine is different from the redox rate for the oxidation of guanine paired with thymine. In particular, the redox rates for guanine oxidation are greater for single-stranded guanine than for guanine paired with adenosine, and for guanine paired with adenosine, greater than for guanine paired with guanine. Guanine is larger than guanine paired with thymine and follows a tendency to be larger than guanine paired with guanine cytosine paired with thymine. Therefore, the method of the present invention is useful for detecting a single base pair mismatch at a predetermined base or a base pair adjacent to the predetermined base.

Advantageously, the difference between the oxidation-reduction kinetics of oxidation of a given base when paired with each of a variety of naturally occurring bases also allows for the identification of bases paired with a given base. I do. The base paired with the predetermined base measures (i) the rate of the detected redox reaction, and (ii) determines the measured reaction rate by using adenosine, cytosine, guanine, or adenosine bound to the predetermined base. By comparing each of the four different known redox kinetics of the DNA with thymine and the oxidizing agent to determine (iii) which of the known kinetics is substantially the same as the measured kinetics; Be identified. The reaction rate is measured according to the technique described above. Similarly, the kinetics of each of four different redox reactions of DNA and oxidizing agents with adenosine, cytosine, guanine, or thymine bound to a given base are the same techniques as those kinetics are known. It is measured according to. The measured reaction rate of the oxidation-reduction reaction of the oxidizing agent with the hybridized DNA is then known as the known oxidation-reduction reaction rate of the oxidizing agent with DNA having adenosine, cytosine, guanine, or thymine bound to a predetermined base. Be compared. For example, a base paired with a given base is determined by determining a known base pairing having a redox reaction rate that is substantially the same as the measured redox reaction rate.

F. DNA Sequencing The present invention provides (a) an oligonucleotide probe comprising a predetermined synthetic base having a unique oxidation potential, contacting a DNA sample with the oligonucleotide probe to form hybridized DNA; ) The oligonucleotide probe has a predetermined number of predetermined synthetic bases and reacts the hybridized DNA with an oxidizing agent such as a transition metal complex that can oxidize the predetermined synthetic base in the oligonucleotide probe by a redox reaction. (C) detecting a redox reaction; (d) measuring a reaction rate of the detected redox reaction; and (e) identifying a base paired with a predetermined synthetic base. Also provided is a method for sequencing DNA comprising the steps of:

According to the methods discussed so far, the DNA sample is amplified before the step of contacting with the oligonucleotide probe according to techniques known to those skilled in the art. Synthetic bases are selected from the group of bases described above and other synthetic bases known to those skilled in the art. The only limitation is that synthetic bases need to have unique oxidation potentials compared to the oxidation potentials of the four naturally occurring bases: adenosine, cytosine, guanine, and thymine. The step of reacting the hybridized DNA with an oxidizing agent, detecting the redox reaction, and contacting the DNA sample with an oligonucleotide probe for measuring the rate of the reaction is performed as described above. The step of identifying the base paired with the given synthetic base comprises: (i) determining the measured reaction rate by using a DNA having adenosine, cytosine, guanine, or thymine bound to the given synthetic base and an oxidizing agent. Comparing to each of two different known redox reaction rates; and (ii) determining substantially the same as the measured reaction rate among the known reaction rates.

In another embodiment, the oligonucleotide probe further comprises a second predetermined synthetic base. The second predetermined synthetic base has a unique oxidation potential different from the oxidation potential of the first predetermined synthetic base. In this embodiment, detecting the redox reaction between the predetermined base and the oxidizing agent further includes detecting a redox reaction between the same second predetermined synthetic base and the oxidizing agent. In addition, the step of measuring the oxidation-reduction kinetics includes measuring the oxidation-reduction kinetics of oxidation with a second predetermined base with a similar oxidizing agent. Further, the step of identifying a base paired with the predetermined synthetic base further comprises the step of identifying a base paired with a similar second predetermined synthetic base. According to this example, the redox reaction of both given bases is detected, and ultimately, the base paired with each given synthetic base is identified using the methods described above. As will be apparent to one of skill in the art, each given synthetic base is distinct from the oxidation potential of all other given synthetic bases and exhibits a unique oxidation potential that is different from the oxidation potential of each of the four naturally occurring bases. In some cases, the above method is performed using two or more predetermined synthetic bases. Not only is each base paired with a given base identified in accordance with the methods described herein, but the DNA is also sufficient to have a given synthetic base at a different site in the DNA sample to identify each base. The sequence is determined by repeating the steps of the above method using different oligonucleotide probes. In other words, the DNA sample contains a sufficient number of synthetic bases, each probe sequence comprising at least one predetermined synthetic base, which differs along the probe sequence in each oligonucleotide probe and is located at the calculated site. Sequenced by providing an oligonucleotide probe. In this method, detection of repetition of a redox reaction of a hybrid DNA with an oxidizing agent, measurement of a redox reaction rate, and identification of a base paired with a predetermined synthetic base include identification of a base-pair base in a sequence of a DNA sample. Bring.

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

Many variations of the above devices are, of course, apparent to one of ordinary skill in the art. Many DNA sample containers are suitable containers known to those skilled in the art, including microtiter plates, test tubes, petri dishes, culture vials, solid supports, etc., that can contain DNA samples. The sample handling means is a suitably designed sample container handling means known to those skilled in the art that can carry a DNA sample container.

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

In another embodiment of the device of the present invention, the device for detecting DNA hybridization comprises: (a) a DNA sample container; (b) an oligonucleotide probe delivery means for delivering a plurality of oligonucleotide probes to the DNA sample container. (C) an oxidizing agent delivery means for delivering an oxidizing agent to a DNA sample container; and (d) an oxidation-reduction reaction detector for detecting an oxidation-reduction reaction. This device is disclosed in U.S. Pat. Nos. 5,143,854 and 5,405,783 to Pirrun et al .; Fodor et al. Nature, 364, p. 555 (1993), the disclosure of which is hereby incorporated by reference in its entirety. Years); Angew. {Chem. No. 107, 356 (1995); and Noble Analytical Chemistry 67 (5), page 21 (1995), adapted for use with fixed probes.

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

Suitable oxidant delivery means for delivering oxidants to DNA sample containers have been described above. A preferred oxidant delivery means comprises a solid support having the oxidant immobilized thereon. According to one embodiment, the oxidizing agent and multiple oligonucleotide probes are immobilized on the same solid support.

The device according to the present invention is useful for performing a diagnostic analysis of changes in a DNA sample. Numerous oligonucleotide probes allow analysis and detection of a variety of DNAs in a single sample, thus providing a useful tool for screening a single sample for changes in DNA, including pathogens and viruses.

H. RNA Hybridization Detection, RNA Sequencing and RNA Mismatch Detection Also disclosed herein are RNA hybridization detection methods, RNA sequencing methods and RNA mismatch detection methods. RNAs useful in performing such methods include, but are not limited to, ribosomal RNA, transfer RNA or pathogenic RNA (eg, RNA obtained from RNA viruses such as retroviruses, HIV-1 and others). The first aspect of the invention therefore comprises (a) contacting the RNA sample with an oligonucleotide probe to form hybridized RNA; and (b) the oligonucleotide probe has at least one predetermined base. Reacting the hybridized RNA with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe in a redox reaction; (c) detecting a redox reaction; and (d). Determining the presence or absence of the hybridizing RNA from the redox reaction detected at the predetermined base.

More specifically, the method for detecting hybridized RNA comprises: (a) contacting an RNA sample with an oligonucleotide probe to form hybridized RNA; and (b) the oligonucleotide probe comprises at least one predetermined base. Reacting the hybridized RNA with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe by a redox reaction; (c) detecting the redox reaction; ) Measuring the rate of the detected redox reaction; (e) comparing the measured rate to the rate of the redox reaction of the transition metal complex with the single-stranded RNA; and (f) measuring Determining whether the rate of the reaction performed is substantially the same as the rate of the redox reaction of the transition metal complex with the single-stranded RNA. .

The method of RNA sequencing comprises the steps of (a) contacting an RNA sample with the oligonucleotide probe to form a hybridized RNA, wherein the oligonucleotide probe comprises a predetermined base having a unique oxidation rate; (b) Reacting the hybridized RNA with a transition metal complex capable of oxidizing a predetermined base in the oligonucleotide probe in a redox reaction, the oligonucleotide probe having a predetermined number of predetermined bases; (c) The method comprises the steps of: detecting a redox reaction; (d) measuring a rate of the detected redox reaction; and (e) identifying a base paired with a predetermined synthetic base. Oligonucleotide probes, hybridization methodology, detection of oxidants, redox reactions, and equipment useful for practicing these methods are substantially set forth in Sections A-H above and are known to those of skill in the art. It applies to the use of RNA as a nucleic acid sample according to the principle (for example, replacing uracil with the base thymine).

I. Detection of Certain Bases on Nucleic Acids of Interest In the methods already specifically described, metal complexes are used to obtain electrochemical currents from single and double stranded DNA or nucleic acids. Certain bases, such as guanine, provide an electrical signal that is much weaker than for double-stranded DNA. Such a method effectively exhibits high structural sensitivity and can identify single base mismatches. Such methods are therefore particularly useful for DNA sequencing. However, two disadvantages of such a method are: (a) the presence of a negative signal in going from the probe strand to the hybrid, and (b) the lack of signal amplification. The following techniques provide solutions to these problems. In addition, the following techniques are particularly useful for diagnostic analysis and for quantitative detection of nucleic acids.

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

The method is performed on a test sample containing the target nucleic acid. Any presumed to contain the nucleic acid of interest, including but not limited to tissue samples such as biopsy samples and biological fluids such as blood, sputum, urine and semen samples, bacterial cultures, soil samples, food samples, etc. Are used. The nucleic acid of interest may be of any origin, including animal, plant, or microbial (eg, viruses, prokaryotic and eucalotic bacteria, protozoa, fungi, protactista, etc.), according to the particular purpose of the test. The sample is processed or purified according to well-known techniques known to those skilled in the art prior to performing the method; if desired, the nucleic acid is digested there, crushed and / or amplified (if desired) before the method is performed. See above).

As schematically shown in FIG. 4, the method comprises the steps of (a) contacting a test sample with an oligonucleotide probe that specifically binds to a nucleic acid of interest to form a hybridizing nucleic acid; Contacting the nucleic acid with a transition metal complex that oxidizes a predetermined base in a redox reaction; (c) detecting the presence or absence of a redox reaction bound to the hybridized nucleic acid; Determining the presence or absence of the target nucleic acid in the test sample from the redox reaction detected with the base. As shown in FIG. 4, the oligonucleotide probe has a separation step that occurs before the detection step (eg, between steps (a) and (b) or between steps (b) and (c)), Immobilized on a solid support to facilitate separation of the test sample from the hybridizing nucleic acids. In addition, oligonucleotide probes can be used in solution and (e.g., by a biotin-avidin binding interaction in which biotin is bound to the oligonucleotide probe and avidin is immobilized on a solid support, e.g., by an agent that binds the oligonucleotide probe. ) Freely provided among other means provided for separating hybridized nucleic acids from a sample.

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

In addition, the oligonucleotide probe must be free of, or at least substantially free of, a given base (ie, a signal that does not interfere with the signal from the probe or that is not mistaken for a signal from the nucleic acid of interest). Or less of the given base). If a sequence of naturally occurring bases that readily hybridize to the nucleic acid of interest is not available, a strategy of using another base that is redox-inactive (discussed below) is used.

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

For example, oligonucleotide probes that do not contain any guanine residues (eg, only A, T, and C) are used. The cyclic voltammogram of Ru (pby) ₃ ²⁺ in the presence of this chain is very similar to that without the oligomer. This strand then contains guanine in either or both the overlapping base pair region and the overhang region, assuming that the nucleic acid of interest is longer than the oligonucleotide probe (indicated in FIG. 4 by a "G" adjacent to the nucleic acid strand of interest). ) Hybridizes with the desired strand. As a large number of guanines are detected, the signal is amplified relative to the number hybridized. When genomic DNA or RNA is the strand of interest, a large number of overhanging guanines are encountered that provide large signal amplification. For example, ribosomal RNA contains as many as 1000 guanines for a particular organism, thus providing approximately 1000-fold amplification for each hybridization result. For example, in one preferred embodiment, the analysis for a given base on the strand of interest comprises a solid surface directed near the electrode surface that gives a low background signal when swept in the presence of the mediator. And immobilization of the upper (preferably non-redox active) probe strand. The solid surface is then contacted with a solution of the desired strand containing a predetermined base. Assuming hybridization occurs, the strand of interest is now in close proximity to the electrode and an increase in current is detected.

Quantification of nucleic acids. The invention is particularly well suited for quantitative detection of nucleic acids. In the cases described in this section, the rate constant for oxidation of the hybrid by an oxidant (eg, Ru (pby) ₃ ²⁺ ) can be determined from a cyclic voltammogram (or other electrical signal) by digital simulation. Under most conditions, in accordance with this reaction is second order kinetics, i.e. the rate = a ^{_{k [Ru (bpy) 3 2+}} ] [DNA], where k is a particular probe - specific for the desired product hybrid such is the rate constant, the concentration of [Ru ^{_(bpy) 3 2+]} oxidizing agent, and [DNA] is the concentration of Hairiddo (may be a DNA-RNA HYBRID). If k and [Ru ^{_(bpy) 3 2+]} is known, it is possible to determine the amount of hybrid. Indeed, a calibration curve for the current gain obtained with different amounts of the standard solution containing the DNA and RNA of interest was constructed and the current gain was used to obtain the amount of hybrid directly. This amount is then directly compared with the amount of the target substance. For example, M. Holdniy et al. Virol. 69, pp. 3510-3516 (1995); See Mellors et al., Science 272, 1167-1170 (1996).

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

J. Redox Inactive Surrogate Bases One disadvantage of the method described in Section H above is that the oligonucleotide probes preferably do not contain a substantial number of certain bases (eg, guanine). A solution to this problem is to replace guanine in the probe strand (ie, a base with another base having a higher binding affinity than that shown in the nucleic acid duplex, such as guanine) and to react with the appropriate reaction. The use of a surrogate base that is not oxidized by the oxidizing agent under the conditions. Examples of such surrogate bases when guanine is the given base are inosine and 7-deaza-guanine.

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

Oligonucleotide probes, methodology for hybridization, methods for oxidizing agents and redox reactions, detection of redox reactions, and equipment useful for performing these methods are as set forth in Sections AI above.

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

Oligonucleotide probes, methodology for hybridization, methods for oxidizing agents and redox reactions, detection of redox reactions, and equipment useful for performing these methods are as set forth in Sections AI above.

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

The method for detecting the presence or absence of a target nucleic acid in a test sample presumably containing the same is as follows: (a) the capture probe specifically binds to the target nucleic acid, and provides an oligonucleotide capture probe. (B) contacting the test sample with a capture probe to form a hybridized nucleic acid; and (c) the signal probe specifically binds to the nucleic acid of interest, generating a hybridized nucleic acid sandwich. Contacting the oligonucleotide signal probe with the hybridizing nucleic acid to oxidize the predetermined base in a redox reaction. Contacting the transition metal complex with the hybridizing nucleic acid. It consists (f) and the detected presence or stage of determining the absence of target nucleic acids in a test sample from the oxidation-reduction reaction at a given base; and standing or detecting the absence. The separation step is between steps (b) and (c), or between steps (c) and (d), wherein the test sample is preferably separated from the capture probe. Depending on the assay format (eg, spontaneous or allogeneic), the oligonucleotide capture probe will hybridize from a solid support (eg, polymeric beads, plates, or the internal surface of a microtiter plate well), or a test sample as described above. It is immobilized on alternative means provided for separating nucleic acids.

Many "sandwich" analysis formats are known. The choice of the type of analysis is not critical, and an appropriate format is used to practice the present invention. For example, an oligonucleotide capture probe is immobilized on a solid support, as described in US Pat. No. 4,486,539 to Ranki et al. As described in U.S. Pat. No. 4,868,104 to Kurn et al., An oligonucleotide probe comprises a polymer forming unit, the polymerization of which separates the sandwich of hybridizing nucleic acids. M. S. Clinical Chem. As described in Vol. 39, pp. 725-726 (1993), the signal probe is straight or branched. A mediator polynucleotide that binds an oligonucleotide capture probe to the immobilized polynucleotide is used, as described in U.S. Patent No. 4,751,177 to Stabinsky. R. Goodson European Patent Application No. 0 238 332: W.C. {Harrison} European Patent Application No. 0 139-489; Oligonucleotide probes can be part of a specific binding pair (eg, biotin) and immobilized on a solid support (eg, avidin), as described in European Patent Application No. 0 {192} 168 to Datatagupta. The second binding interaction with the other part of the pair is linked to a sandwich of hybridized nucleic acids separated from the test sample.

Oligonucleotide probes, methodology for hybridization, methods for oxidizing agents and redox reactions, detection of redox reactions, and equipment useful for performing these methods are as set forth in Sections AK above.

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

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

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

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

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

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

Oligonucleotide probes as described in connection with Formulas I and II above are made according to known techniques and are modified in the disclosure of the examples which follow, as will be readily apparent to those skilled in the art. .

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

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

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

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

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

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

The following examples are provided to illustrate the invention and should not be construed as limiting. In these examples, cm ² / s means square centimeters per second, M means molarity, M ⁻¹ s ⁻¹ means moles per second, eV means electron volts, V means volts, nm means nanometers, GMP means guanine 5'-monophosphate, ITO means tin-doped indium oxide electrode. Cyclic voltammograms were obtained using EG + G Princeton Applied Research Potentiostat / Galvanostat model 273A according to known techniques. The ITO working electrode is available from Delta Technology, Inc., 13960 13th North, Stillwater, 55082-1234, Minnesota, USA. Ltd. Made from ITO coated soda lime glass sheet, part number CH-501N-1514, available from S.A. Nylon membranes are available from Amersham Corp. of Clearbrook Drive 2636, Arlington Heights, 60005, Illinois, USA. Is available as a high bond N + (HYBOND-N +) nylon membrane with catalog number RPN 1210B.

Ru (bpy) with contact enhancement produced by multiple turnover of oxidation of DNA by the oxidized form of the metal complex to be observed during the measurement a single voltammetric sweep cyclic voltammogram ₃ ^2+, the cyclic voltammogram of Ru ^{_(bpy) 3 2+} with and without Ushisaimosu DNA is shown in Figure 1. In FIG. 1, the uniform line represents a scan of 50 μM Ru (bpy) ₃ ²⁺ at 25 mV / s in 700 mM NaCl / 50 mM sodium phosphate buffer, the dashed line represents 50 μM Ru (bpy) ₃ ²⁺ 1 represents the voltammogram of 3.0 mM (nucleotide) bovine thymos DNA. Since the diffusion coefficient of DNA (ie, 2.0 × 10 ⁻⁷ cm ² / s) is much smaller than that of the metal complex (8.0 × 10 ⁻⁶ cm ² / s), the voltammetry of the DNA-bound redox pair is It should be analyzed by a square diagram for the combined and uncombined forms. This phenomenon generally results in a dramatically reduced current for the bound form; however, at sufficiently high ionic strength ([Na ⁺ = 0.8 M]), the binding of the metal complex affects the current response because Is too weak. In this case, the current is analyzed by a simple EC 'mechanism.

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

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

Rate constant for oxidation of DNA by Ru ^{_(bpy) 3 3+} was confirmed by two separate experiments. First, the square wave voltammogram was used to obtain pseudo-first order kobs for Equation 2 by fitting with the COOL ^TM algorithm. COOLTM algorithm is to use a very different fitting approach and DIGSIMTM, however, the plot of _{k obs} for DNA is linear, consistent with the rate constant obtained from fitting cyclic voltammograms with DIGSIM ^TM, the secondary The rate constant k = 8.2 × 10 ³ M ⁻¹ s ⁻¹ was given. Secondly th, Ru (bpy) ₃ ³⁺ reference samples were prepared and reacted with DNA directly in a rapid scan was stopped with the flow. An overall analysis of the time-dependent spectra between 350 and 600 nm shows that Ru (bpy) ₃ ³⁺ converts cleanly to Ru (bpy) ₃ ²⁺ with a rate constant of 12 × 10 ³ M ⁻¹ s ⁻¹ without intermediates. It was shown to be. Thus, Ru (bpy) ₃ rate constant of DNA oxidation by ^3+, dramatically different by two independent electrochemical measurements using fitting protocols and using fitting full visible spectrum non-electrochemical stopped-flow technique Was clearly established by

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

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

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

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

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

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

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

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

Modified base avoiding oxidation in the probe strand: 7-deaza-guanine This example is provided except that 7-deaza-guanine is used as a modified base instead of guanine to provide a redox-inactive probe strand. , In substantially the same manner as in Example 4 above.

7-Deaza-guanine is oxidized at a rate of only 10 ³ M ⁻¹ s ⁻¹ , which is about two orders of magnitude slower than guanine and slow enough to provide a redox-inactive probe chain.

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

For experiments to obtain only the cyclic voltammogram of the metal complex, the ITO electrode is first prepared with a buffer. The nylon disc (without DNA) is then inserted into the electrochemical cell and 200 μL of a 200 μM metal complex solution is pipetted into the cell. To Os ^{_(bpy) 3 2+} experiments, prior to electrochemical analysis, equilibration time of 6 minutes was used. To Ru ^{_(bpy) 3 2+} experiments, prior to electrochemical analysis, was employed for 15 minutes equilibration time. Cyclic voltammograms were acquired using a PAR 273A potentiostat at a sweep rate of 25 mM / s.

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

The cyclic voltammogram of Ru ^{_(bpy) 3 2+} in nylon -ITO electrode is shown in Figure 11. The dotted line shows the voltammogram when the nylon membrane was immersed in bovine thymos DNA before it was attached to the electrode. There is a large contact current on the DNA-labeled membrane, corresponding to that observed in solution. The experiment shows that it is not necessary for the diffusion of that and DNA Ru (bpy) ₃ ²⁺ is free to diffuse in a nylon film to achieve a contact current. FIG. 11 also shows that higher contact currents are observed at lower salt concentrations due to the increased interaction between the mediator and the immobilized DNA.

Figure 12A shows the same experiment using Os ^{_(bpy) 3 2+} as mediator. The osmium complex does not oxidize guanine, and thus the current increase observed in the presence of guanine must result from preconcentration of mediators by DNA bond formation. In fact, current for Os ^{_(bpy) 3 2+} is more in the presence of DNA at the nylon electrode is lower than the DNA absence. Experiments, DNA is due to the catalytic reaction of increased current is exclusively owned for Ru (bpy) ₃ ²⁺ when bound to nylon electrodes, it does not depend on the difference in the trivalent bond formation. The effect of salt concentration is shown in FIG. 12B and is small compared to the effect of the large salt observed on the catalytic reaction.

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

Detection of RNA Bound to Nylon Membrane Attached to ITO Electrode An experiment was described in Example 6 except that tRNA from Bakers Yeast (obtained from Sigma) was used instead of bovine thymos DNA. It is carried out as described above. Nylon film disks were dipped in a solution of tRNA as described in Example 6.

Ru ^{_(bpy) 3 2+} cyclic voltammetry in the presence is shown in Figure 13. As with DNA, the contact current is observed with both buffers and is greater with lower concentrations of salt. The difference observed in the current between the high and low salt concentrations is not as dramatic as that observed with the DNA of Example 6, since tRNA does not bind cations like DNA, so the salt effect is less dramatic. is not.

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

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

For RNA mismatch detection, a DNA (or RNA, PNA, or other alternative) probe is immobilized on a solid support. Certain bases in the probe strand are more easily oxidized than other bases. Surface is contacted with a solution of interest RNA, is then contacted with a solution of Ru ^{_(bpy) 3 2+} or other mediators. The result of hybridization (perfect, unpaired, or mismatched) is then determined at the given base in the same manner as for DNA.

The method for detecting the predetermined base sequence is carried out as described in Example 3. The four cyclic voltammograms placed in FIG. 14 show that the current with 5′-G is much smaller than for 5′-GG, which is much smaller than for 5′-GGG. A very large increase in this current is observed for both single-stranded and double-stranded containing GG and GGG sequences. As shown in FIG. 15, the increase in current due to the addition of G to the same chain is much smaller if the Gs are staggered, so the increase in current is not simply due to an increase in the number of Gs. Since the 5'-G of GGG is much more easily oxidized than a single G, it is possible to select a mediator (with a lower redox potential) that can oxidize GGG instead of G.

Ru (4,4'-dimethyl - _bipyridine) cyclic voltammogram of ^{3 2+} is shown in Figure 16 along with repeat scans in the presence of a single G oligonucleotide and GGG oligonucleotide. As shown, contact current is only observed in the presence of the GGG oligonucleotide. This example demonstrates the ability to modulate the potential of mediators so that more easily oxidized sequences can be detected in the presence of background guanine. The same scheme can be applied to detecting a single synthetic base derived to make it more easily oxidized than guanine.

The example shows that it is possible to lower the potential of the mediator and to identify bases or base sequences that can be more easily oxidized.

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

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

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

The results of four cyclic voltammograms are shown in FIG. 17, where curve A is Ru (4,4′-Me ₂ -bpy) ₃ ²⁺ alone (4,4′-Me ₂ -bpy = 4,4′-dimethyl) -Bipyridine). According addition of _5'-GMP, Ru increase of (Me 2 ^{_bpy) 3 2+} wave is not observed; however the addition dramatic increase of current but 6-mercaptopurine guanosine 5'-monophosphate (6-S-GMP) Lead (curve B). The peak current in the presence of 5'-GMP is identical to that of curve A. The data shows that detection of 6-mercaptoguanosine base in the presence of background natural guanine is possible.

Detection of DNA Hybridization with Predetermined Base on Target Chain Nylon membrane (Hybond N +, Amersham, 480-600 μg / cm ² ) is cut into a circular shape with a diameter of about 6 mm. The nylon disc is placed in a concentrated solution of polycitidic acid (available from Sigma) in water and soaked for one hour. The disc is then removed from the polycitidic acid (poly [C]) solution, placed on parafilm and dried. As the disc dries, an additional 15 μl of poly [C] solution is added to the film in three 5 μl aliquots. The disc is completely dried. The dried nylon disc is then subjected to a low concentration of salt buffer (50 mM Na-phosphate, pH = 6.8, [Na ⁺ ]) to remove poly [C], which is not strongly bound during the dipping step. (80 mM).

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

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

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

The cyclic voltammogram is reported in FIG. The probe sequence poly [C] is immobilized on a Hybond N + nylon membrane and the hybridization protocol is performed in buffer ("pseudohybridization"). Film is attached to the ITO working electrode, a cyclic voltammogram of Ru ^{_(bpy) 3 2+} is obtained (A). The membrane is immersed in a solution of poly [G] and hybridization is performed according to the same protocol. Ru (bpy) ₃ cyclic voltammogram ²⁺ is then measured (B), and a large current enhancement is obtained due to the catalytic oxidation of hybridized poly [G] target. As shown in FIG. 19, the analysis is specific for well-defined sequences. FIG. 19 compares voltammetry on poly [C] membranes when the hybridization process is performed in buffer (A) or in a solution of single-stranded bovine thymos DNA (B). FIG. 19 shows that no current increase is obtained if the target sequence is not present.

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

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

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

It is a graph showing associated with Ushisaimosu DNA and the cyclic voltammograms of Ru ^{_(bpy) 3 2+} without. Is a graph showing cyclic voltammograms of Ru ^{_(bpy) 3 2+} in the presence of 5'-AAATATAGTATAAAA as single chain (C) and those hybridized to a complementary strand (A & B). 1 is a schematic diagram of one illustrative apparatus useful for performing the method of the present invention. FIG. 3 is a schematic diagram of a detection method particularly useful for quantitative detection of DNA in which a predetermined base is placed on a target nucleic acid. of pH 7, sodium phosphate buffer at 50mM containing NaCl of 0.7M, it is a graph showing cyclic voltammograms of ^{_{Ru (bpy) 3 2+ (25μM}} ) at a sweep rate of 25 mV / s. Structure of inosine and guanine are shown in the ^{_{figure, (A) Ru (bpy)}} 3 2+ (25μM), Ru with (B) an inosine 5'-monophosphate ^{_{(0.3mM) (bpy) 3 2+}} (25μM ), and it is a graph showing a cyclic voltammogram (C) Ru with guanosine 5'-monophosphate ^{_{(0.3mM) (bpy) 3 2+}} (25μM). FIG. 5 is a schematic diagram of an alternative embodiment of the invention of FIG. 4 wherein a given base is on the extension product of a terminal transferase. FIG. 5 is a schematic view of an alternative embodiment of the invention of FIG. 4 implemented in a sandwich analysis format. FIG. 3 is a top view schematic diagram of a micropotential device useful for performing the method of the present invention. FIG. 10 is a side view of a portion of the apparatus shown in FIG. 9. _Ru in buffer dipped nylon ^{(bpy) 3 2+ (200μM)} , Ru (bpy) 3 2+ in the DNA absorption nylon high salt (700 mM added NaCl) of buffer (200 [mu] M), and low salt (i.e., NaCl not added) is a graph showing _Ru in DNA absorption nylon buffer ^(bpy) 3 2+ in (200 [mu] M), the cyclic voltammograms using nylon improved ITO electrodes. Is a cyclic voltammogram of _Os using nylon improved ITO electrode immersed in or in DNA in buffer ^{(bpy) 3 2+ (200μM)} , a graph showing a cyclic voltammogram with the added NaCl to 700 mM. Is a cyclic voltammogram of _Os using soaked nylon improved ITO electrode or during DNA in buffer ^{(bpy) 3 2+ (200μM)} , a graph showing a cyclic voltammogram of NaCl was not added. _Ru in buffer dipped nylon ^{(bpy) 3 2+ (200μM)} , Ru (bpy) 3 2+ in the tRNA absorption nylon high salt (700 mM added NaCl) of buffer (200 [mu] M), and low salt (i.e., NaCl not added) is a graph showing a cyclic voltammogram at _{which Ru} ^(bpy) 3 2+ illustrating the cyclic voltammograms (200 [mu] M), nylon improved ITO electrodes in tRNA absorption nylon buffer. Ru (bpy) ₃ ²⁺ (25 μM) alone with a sweep rate of 25 mV / s and 5′-AAATATAGn TATAAAA (100 μM in the chain) with n = 1 (G), 2 (GG), or 3 (GGG) It is a graph which shows the cyclic voltammogram of the accompanying. Cyclic voltammograms of Ru (bpy) ₃ ²⁺ (25 μM) alone with a sweep rate of 25 mV / s and 5′-AAATAT (AGT) nATAAAAA (100 μM in the chain) with n = 1, 2, or 3 are shown. It is a graph shown. Sweep rate is 25 mV / s, 25 [mu] M ruthenium _{(4,4'-dimethyl-bipyridine)} ^{3 2+} (or _{"Ru (4,4'-Me 2 -bpy} ) 3 2+") alone (uniform) and 5 ' FIG. 2 is a graph showing cyclic voltammograms of those with (AAATATAGTATAAAAA (dot) and 5′-AAATATAGGGTATAAAAA (dash) (100 μM in the chain). Curve (A) is a single Ru _(4,4'-Me 2 _-bpy) represents ^{3 2+,} curve (B) is Ru in the presence of 6-mercaptoguanosine 5'-monophosphate of 0.70 mm (4 , 4′-Me ₂ -bpy) ₃ ²⁺ , 0.2 mM Ru (4,4) in 50 mM sodium phosphate buffer (pH 7) with 0.7 M NaCl at a sweep rate of 25 mV / s. _4'-Me 2 _-bpy) is a graph showing a cyclic voltammogram of ^{3 2+.} The membrane was impregnated with poly [C] and subjected to a hybridization protocol in a buffer solution (A) and in a concentrated solution of poly [G] (B) at 200 μM on an ITO working electrode fitted with a Hybond N + nylon membrane. is a graph showing cyclic voltammograms of ru ^{_(bpy) 3 2+.} The membrane is impregnated with poly [C] and undergoes a hybridization protocol in buffer [A] and in a concentrated solution of denatured bovine thymos DNA. 200 μM Ru (bpy) at an ITO working electrode fitted with a Hybond N + nylon membrane. it is a graph showing a cyclic voltammogram of ₃ ^2+. Cyclic voltammogram of 200 μM Ru (bpy) ₃ ²⁺ after DNA absorption on a nylon-modified glassy electrode without DNA (A) or its nylon film (sweep rate = 25 mV / s) FIG.

Claims (27)

A microelectronic board,
A conductive redox detection electrode on the substrate,
A first element of a specific binding pair adjacent to the detection electrode and immobilized on a non-conductive layer, the first element being capable of reacting with a second element of the specific binding pair present in the sample; And consists of
A microelectronic device useful for electrochemical detection of elements of a binding pair, capable of detecting an oxidation-reduction reaction generated when a potential is applied to the detection electrode. The microelectronic device according to claim 1, wherein the microelectronic substrate has a sample container, and the container has the conductive redox detection electrode and the immobilized first element. The microelectronic device according to claim 2, wherein the microelectronic substrate includes the container having a plurality of conductive oxidation-reduction detection electrodes and a plurality of immobilized first elements. The microelectronic device according to claim 3, further comprising a conductive reference electrode having a conductive metal. The microelectronic device according to claim 3, further comprising a conductive auxiliary electrode having a conductive metal. The microelectronic device according to claim 5, wherein the oxidation-reduction reaction is detectable via an electrical connection from each conductive oxidation-reduction detection electrode. 7. The microelectronic device according to claim 6, wherein any one of the plurality of first elements of the specific binding pair is the same as or different from any other one of the plurality of first elements. The microelectronic substrate according to claim 7, wherein each element in the plurality of first elements of the specific binding pair is different from other elements in the plurality of first elements. The microelectronic device according to claim 6, wherein the first element is selected from the group consisting of nucleic acids and proteins. 10. The microelectronic device of claim 9, wherein the first member of the specific binding pair comprises an oligonucleotide capture probe, wherein the oligonucleotide capture probe is between 4 and 100 nucleotides in length. 7. The microelectronic device according to claim 6, further comprising an oxidation-reduction reaction detector. 7. The microelectronic device according to claim 6, wherein the substrate is silicon. The microelectronic substrate according to claim 6, wherein the substrate is glass. 7. The microelectronic device according to claim 6, wherein the microelectronic device comprises one or more sample containers. 15. The microelectronic device of claim 14, wherein the plurality of first elements of the specific binding pair in any sample container are the same or different than the plurality of first elements in any other sample container. . The microelectronic device according to claim 15, wherein the plurality of first elements of the specific binding pair in each sample container are the same as the plurality of first elements in other sample containers. The microelectronic device according to claim 14, wherein any one of the plurality of first elements in the specific sample container is the same as or different from any other element in the specific sample container. 18. The microelectronic device of claim 17, wherein each of the plurality of first elements in a particular sample container is different from any other element in the particular sample container. The microelectronic device according to claim 14, wherein each sample container includes a microsubstrate, a conductive redox detection electrode, and a first element for immobilizing a specific binding pair. The microelectronic device according to claim 14, wherein the first element is selected from the group consisting of nucleic acids and proteins. 15. The microelectronic device of claim 14, wherein the first member of the specific binding pair comprises an oligonucleotide capture probe, wherein the oligonucleotide capture probe is between 4 and 100 nucleotides in length. The microelectronic device according to claim 14, further comprising a redox reaction detector. The microelectronic device according to claim 14, wherein the substrate is silicon. The microelectronic device according to claim 14, wherein the substrate is glass. Each conductive redox detection electrode is electrically connected to a potentiostat, the sample container has a sample and an electrolyte solution, each detection electrode is in electrochemical contact with the solution, and the potential is 17. The microelectronic device according to claim 16, comprising an electrochemical medium capable of participating in the electrode and the redox electrode under conditions applied to the electrode. Further comprising a redox reaction detector for detecting the redox reaction, wherein when the sample contains the second member of the specific binding pair, the sample does not contain the second member of the specific binding pair; The microelectronic device of claim 25, wherein the current is more detectable. The microelectronic device includes a plurality of sample vessels, each of which comprises an electrolyte solution, the solution having the conductive redox sensing electrode in electrochemical contact, and the solution having an electrochemical medium. The microelectronic device according to claim 25.

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