MXPA01008897A - Electrochemical detection of nucleic acid hybridization - Google Patents

Abstract

A method of detecting a nucleic acid (e.g., DNA, RNA) that contains at least one preselected base (e.g., adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine) comprises:(a) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction;(b) detecting the oxidation-reduction reaction;and (c) determining the presence or absence of the nucleic acid from the detected oxidation-reduction reaction at the preselectedbase. The method may be used in a variety of applications, including DNA sequencing, diagnostic assays, and quantitative analysis.

Description

ELECTROCHEMICAL DETECTION OF HYBRIDIZATION OF NUCLEIC ACID FIELD OF THE INVENTION The present invention relates to the hybridization and sequencing of the nucleic acid, and particularly to methods for qualitatively and quantitatively detecting the hybridization of the nucleic acid and nucleic acid sequencing methods.

BACKGROUND OF THE INVENTION The detection of individual DNA sequences in heterogeneous DNA samples provides a basis for identifying genes, DNA profiles and novel approaches to DNA sequencing. One approach for the detection of DNA hybridization includes the use of surface-bound DNA sequences that can be tested using an analytical response that indicates hybridization of the oligomer bound at the surface to a sequence in the heterogeneous sample. These analytical methods generally include laser-induced fluorescence that originates from a label covalently fixed on the target DNA strand, which is not sensitive to mismatches of a base in the duplex attached to the surface. For example, the patents of E.U.A. Nos. 5,143,854 and 5,405,783 to Pirrung et al., Fodor, et al., Nature 364: 555 (1993); Bains, Agew. Chem.107: 356 (1995) and Noble, Analitical Chemistry 67 (5): 201A (1995) propose surfaces or "fragments" for this application. In an alternative method, proposed by Hall, et al., Biochem and olec. Bio. inter. 32 (1): 21 (1994), DNA hybridization is detected by an electrochemical method that includes observing the redox behavior of a single-stranded DNA compared to a double-stranded DNA. This technique is also not sensitive to mismatches of a base in the DNA sample. The techniques for detecting mismatches of a base include studies of the chemical or enzymatic cut such as those proposed in the patent of E.U.A. No. 5,194,372 to Nagai et al. However, these techniques are disadvantageous since they require more time and separation technology. The patent of E.U.A. No. 5,312,527 to Mikkelson et al., Discloses a selective voltammetric sequence sensor for detecting target nucleic acid, in which a double-stranded nucleic acid is contacted with a redox-active complex. The complex binds not specifically to double-stranded DNA. Because the complex itself is the redox-active compound that provides a voltammetric signal, the complex does not work in a catalytic manner. The patent of E.U.A. No. 4,840,893 to Hill et al., Discloses an electrochemical test for nucleic acids, in which a competitive ligation event between a ligand and an antiligand is in turn detected electrochemically.

Accordingly, a need remains in the art for a method for detecting DNA hybridization, which includes a method for detecting mismatches of a base, which is both rapid and sensitive, and which can be applied rapidly online.

BRIEF DESCRIPTION OF THE INVENTION In general, the present invention provides a method for detecting a nucleic acid containing at least one preselected base (e.g., adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine and 8-oxo-adenine). The method comprises (a) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; (b) detecting the oxidation-reduction reaction; and (c) determining the presence or absence of the nucleic acid from the oxidation-reduction reaction detected in the preselected base. Depending on the particular mode of the method, and the particular object desired, the method may optionally include the step of making contact between the nucleic acid and a complementary nucleic acid to form a hybridized nucleic acid. As a first aspect, the present invention provides a method for detecting DNA hybridization. The method includes (a) making contact between a DNA sample and an oligonucleotide probe to form a hydrided DNA, (b) reacting the hydrided DNA with a transition metal complex capable of oxidizing a preselected base in the oligonucleotide probe in an oxidation-reduction reaction, wherein the oligonudeotide probe has at least one of the bases pre-selected, (c) detecting the oxidation-reduction reaction and (d) determining the presence or absence of hydrated DNA from the oxidation-reduction reaction detected in the preselected base. As will be mentioned in detail below, the step of detecting the oxidation-reduction reaction can be carried out, in general, by measuring the electron flow from the preselected base. As a second aspect, the present invention provides another method for detecting DNA hybridization. The method includes (a) making contact between a DNA sample and an oligonucleotide probe to form a hybridized DNA, (b) bringing the hybridized DNA into reaction with a transition metal complex capable of oxidizing a preselected base in the DNA probe. oligonudeotide in an oxidation-reduction reaction, wherein the oligonucleotide probe has at least one of the preselected bases, (c) detecting the oxidation-reduction reaction, (d) measuring the reaction rate of the oxidation-reaction. detected reduction, (e) compare the measured reaction velocity with the oxidation-reduction reaction rate of the transition metal complex with a DNA of a chain and then (f) determine whether the measured reaction velocity is essentially the same as the oxidation-reduction reaction rate of the transidon metal complex with DNA from a chain.

As a third aspect, the present invention provides an apparatus for detecting DNA hybridization. The apparatus includes (a) a plurality of DNA sample containers, (b) means for handling samples that carry the plurality of DNA sample containers, (c) an oligonucleotide probe delivery means for delivering the probe of oligonucleotide to each of the DNA sample containers, (d) a transition metal complex release means to release the transition metal complex to each of the plurality of DNA sample containers, and (e) ) an oxidation-reduction reaction detector to detect an oxidation-reduction reaction. As a fourth aspect, the present invention provides a second apparatus for detecting DNA hybridization. The apparatus includes (a) a DNA sample container, (b) an oligonucleotide probe delivery means for delivering a plurality of oligonucleotide probes to the DNA sample container, (c) a metal complex release medium. of transition to release the transition metal complex in the DNA sample container; and (d) an oxidation-reduction reaction detector to detect an oxidation-reduction reaction. As a fifth aspect, the present invention provides a method of DNA sequencing. The method includes (a) contacting between a DNA sample and an oligonucleotide probe to form a hybridized DNA, wherein the oligonucleotide probe includes a preselected synthetic base having a unique oxidation potential, (b) reacting the DNA hybridized with a transition metal complex capable of oxidizing the preselected synthetic base in the oligonucleotide probe in an oxidation-reduction reaction, wherein the oligonucleotide probe has a predetermined number of the preselected synthetic bases, (c) detecting the oxidation-reduction reaction, (d) measuring the reaction rate of the oxidation-reduction reaction detected, and (e) identifying the base paired with the preselected synthetic base. The foregoing and other aspects of the present invention are explained in detail in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the cyclic voltammograms of Ru (bpy) 32+ with and without calf thymus DNA. The solid line represents the scrutiny of 50μM Ru (bpy) 32+ at 25 mV / s in 700 mM NaCl / 50 mM sodium phosphate buffer. The dotted line represents the voltammogram of 50μM Ru (bpy) 32+ and 3.0mM of calf thymus DNA (nudeotide). Figure 2 shows the cyclic valtamograms of Ru (bpy) 32+ in the presence of S'-AAATATAGTATAAAA as a single strand (C) and hybridized to complementary strands (A and B). The speed of scrutiny is 25 mV / s. (A) represents 25 μM of Ru (bpy) 32+ + 100 μM of fully double-stranded DNA (in guanine nudeotides) (5'-AAATATAGTATAAAA) (3'-T GATATCATATTTT). (B) represents Ru (bpy) 32+ with a duplex containing a GA mismatch (5'-AAATATAGTATAAAA) - (3'-TTTATATAATAT? T), and (C) represents Ru (bpy) 32+, a sendlla chain containing a guanine nucleotide (5'-AAATATAGTATAAAA). Figure 3 is a schematic illustration of an illustrative apparatus that is useful for carrying out the methods of the present invention. Figure 4 is a schematic illustration of a particularly advantageous detection method for the quantitative detection of DNA, wherein the preselected base is located on the target nucleic acid. Figure 5 shows the cyclic voltammograms of Ru (bpy) 32+ (25 μM) at a scanning speed of 25 mV / s in 50 mM sodium phosphate pH regulator with 0.7 M NaCl, pH 7. (A ) There is no added nucleotide. (B) With 75 μM of d VTTTTTATACTATATTT]. (C) With 75 μM of the oligomer hybrid of B and d [5'-GGGAAATATAGTATAAAAGGGl. Working electrode: indium oxide contaminated with tin. Eledrodo reference: Ag / AgCI. Counter-electrode: Pt wire. The secondary structure of the hybrid of C is indicated in the figure. Figure 6 shows the cyclic voltammograms of (A) Ru (bpy) 32+ (25 μM), (B) Ru (bpy) 32+ (25 μM) with 5'-inosine monophosphate (0.3 mM), and (C) Ru (bpy) 32+ (25 μM) with 5 * -guanosine monophosphate. The structures of inosine and guanine are shown in the figure.

Figure 7 illustrates schematically an alternative embodiment of the invention of Figure 4, wherein the preselected bases are on an elongation product of the terminal transferase. Figure 8 illustrates schematically an alternative embodiment of the invention of Figure 4, carried out in a sandwich test format. Figure 9 is a schematic top plan view illustration of a microelectronic device useful for carrying out the methods of the present invention; Figure 10 is a side sectional view of a portion of the device illustrated in Figure 9. Figure 11 shows the cyto-voltammograms using ITO electrodes modified with nylon, Ru (bpy) 3 + (200 μM) in nylon soaked with DNA in high salt pH regulator (700 mM NaCl added), and Ru (bpy) 3 + (200 μM) in nylon soaked with DNA in low salt pH regulator (ie, no NaCl was added). Figure 12 shows the cyclic voltammograms of Os (bpy) 32+ (200 μM) using nylon-modified ITO eledrodes soaked with pH or DNA buffer. Figure 12A shows the cyclic voltammogram with 700 mM NaCl added. Figure 12B shows the cyclic voltammogram without NaCI added. Figure 13 shows the cyclic voltammograms in nylon-modified ITO electrodes showing cyclic voltammograms of Ru (bpy) 32+ (200 μM) in nylon soaked with pH regulator, Ru (bpy) 32+ (200 μM) in nylon soaked with TRNA in high salt (700 mM NaCl added), and Ru (bpy) 32+ (200 μM) in nylon soaked with tRNA in low salt buffer (without added NaCl). Figure 14 shows the cyclic voltammogram of Ru (bpy) 3 + (25 μM) alone and with (100 μM in chains) of 5'-AAATATAGnTATAAAA where n = 1 (G), 2 (GG), or 3 (GGG ). The speed of scrutiny is 25 mV / s. Figure 15 shows the cyclic voltammogram of Ru (bpy) 3 + (25 μM) alone and with (100 μM in chains) of S'AAATATfAGTJnATAAAA where n = 1, 2, or 3. The scanning speed is 25 mV / s. Figure 16 shows the cyclic voltammogram of 25 μM of 4,4'-dimethylbipyridine) 32+ of ruthenium (or "Ru (4,4-Me2-bpy) 32+") alone (solid) and with (100 μM in chains) of 5'-AAATATAGTATAAAA (dotted) and 5'- AAATATAGGGTATAAAA (in dashes). The speed of scrutiny is 25 mV / s. Figure 17 shows the cyclic voltammogram of 0.20 mM Ru (4,4'-Me-2-bpy) 32+ in 50 mM sodium phosphate buffer (pH 7) with NaCl of 0.7 M at a scanning speed of 25 mV / s. Curve (A) represents Ru (4,4'-Me2-bpy) 32+ alone. Curve (B) represents Ru (4,4'-Me2 ~ bpy) 32+ in the presence of 0.70 mM 6'-monop- phosphate 5'-monophosphate. Figure 18 shows cyclic voltammograms of 200 μM Ru (bpy) 32+ in tTO working electrodes to which a Hybond N + nylon membrane is fixed. The membranes are impregnated with poly [C] and subjected to the hybridization protocol in pH regulator (A) and a concentrated solution of poly [G] (B). Figure 19 shows cyclic voltammograms of 200 μM of Ru pyJs2"1" in ITO working electrodes to which a Hybond N + nylon membrane is fixed. The membranes are impregnated with poly [C] and subjected to the hybridization protocol in pH regulator (A) and a concentrated solution of denatured calf thymus DNA (B). Figure 20 shows the cyclic voltammograms (scanning speed = 25 mV / s) of 200 μM Ru (bpy) 32+ in a glassy carbon dioxide modified with nylon (A) without DNA or (B) after the adsordion of DNA to the nylon film.

DETAILED DESCRIPTION OF THE INVENTION The term "nudeic acid" is used herein to refer to any nucleic acid, including both DNA and RNA. The nucleic acids of the present invention are typically polynucleic acids; that is, individual nucleotide polymers that are covalently linked by 3 ', 5 * phosphodiester linkages. The term "complementary nude acid" is used herein to refer to any nudeic acid, including oligonucleotide probes that specifically bind to another nucleic acid to form a hybridized nudeic acid.

The phrase "determine the presence or absence of" is designed to include both the qualitative determination and the quantitative determination of the presence or absence of the detected event (eg, DNA hybridization, RNA hybridization, target nucleic acid detection, etc.). The terms "hybridized DNA" and "hybridized nucleic acid" refer to a single-stranded DNA that is hybridized to form a DNA or double-stranded nucleic acid that is hybridized to form a DNA or triple-helical nucleic acid. Although the methods and apparatus of the present invention are sometimes explained with respect to the DNA of the present, this is for purposes of clarity and it is to be understood that the methods and apparatus of the present invention can be applied to other nucleic acids such as RNA A. Nucleic Acid Amplification Methods Since the methods of the present invention include contacting a DNA sample with an oligonucleotide probe to produce a hybridized DNA, it may be desirable for ain applications to amplify the DNA before making contact with the DNA. the probe. The amplification of a selected or target nucleic acid sequence can be carried out by any suitable means. See generally D. Kwoh and R. Kwoh, Am. Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplification techniques include, but are not limited to, the polymerase chain reaction (including, for RNA amplification, the reverse transcriptase polymerase chain reaction), the ligase chain reaction, the amplification of chain shift, amplification on the basis of transcription (see D. Kwoh et al., Proc. Nati. Acad Sci. USA 86, 1173-1177 (1989)), self-sustained sequence replication (or "3SR") (see J. Guatelli et al., Proc. Nati, Acad. Sci USA 87, 1874-1878 (1990)), the Qβ replicase system (see P. Lizardi et al., Biotechnology 6, 1197-1202 (1988)), the amplification based on nucleic acid sequences (or "NASBA") (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), repair chain reaction (or "RCR") (see R. Lewis, supra), and boomerang DNA amplification (or "BDA") (see R. Lewis, supra). The bases incorporated in the amplification product can be natural or modified bases (modified before or after the amplification), and the bases can be selected to optimize subsequent electrochemical detection steps. Polymerase chain reaction (PCR) can also be carried out according to known techniques. See, e.g., the patents of E.U.A. No. 4,683,195; 4,683,202; 4,800,159 and 4,965,188 (the description of all references to US Patents cited herein are incorporated herein by way of reference). In general, PCR includes, first, treating a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) with an oligonucleotide primer for each strand of the specific sequence that will be detected under hybridization so that one extension product of each inventor is synthesized and is complementary to each nucleic acid strand, with the inidators sufficiently complementary to each strand of the specific sequence to hybridize therewith so that the extension product synthesized from each initiator, when separated from its complement, can serve as a pattern for the synthesis of the extension product of the other initiator, and then treating the sample under denaturing conditions to separate the initiator extension outputs from their patterns if the sequence or sequences that will be detected are present. These steps are repeated cyclically until the desired degree of amplification is obtained. Detection of the amplified sequence can be carried out by adding to the reaction product an oligonucleotide probe capable of hybridizing to the reaction product (e.g., an oligonucleotide probe of the present invention), the probe carrying a detectable label, and then detecting the label according to convenient techniques. Where the nucleic acid to be amplified is RNA, the amplification can be carried out by the initial conversion of the DNA by reverse transcriptase according to known techniques. The chain shift amplification (SDA) can be carried out according to known techniques. See generally G. Walker et al., Proc. Nati Acad. Sd. E.U.A. 89, 392-396 (1992); G. Walker et al., Nucleic Acids Res. 20, 1691-1696 (1992). For example, the SDA can be carried out with a single amplifier or a pair of amplification initiators, achieving exponential amplification with the latter. In general, the primers of the SDA amplification comprise, in the 5 'to 3' direction, a flanking sequence (whose DNA sequence is not critical), a restriction site for the restriction enzyme used in the reaction, and a oligonucleotide sequence (e.g., an oligonudeotide probe of the present invention) that hybridizes to the target sequence that will be amplified and / or detected. The flanking sequence, which serves to fuse the ligation of the restriction enzyme to the recognition site and which provides a DNA polymerase initiation site after the restriction site has been cut, preferably has from about 15 to 20 nucleotides. of length; the restriction site is functional in the SDA reaction (ie, the phosphorothioate linkages incorporated in the initiator chain do not inhibit subsequent cleavage with a condition that can be met through the use of a nopalindromic recognition site); the portion of the oligonucleotide probe is preferably about 13 to 15 nucleotides in length. The ligase chain reaction (LCR) is also carried out according to conventional techniques. See, e.g., R. Weiss, Science 254, 1292 (1991). In general, the reaction is carried out with two pairs of oligonucleotide probes: a pair is linked to a chain of the sequence that will be arrested; the other pair is linked to the other chain of the sequence that will be stopped. Each pair together completely overlaps the chain to which it corresponds. The reaction is carried out, first, by denaturing (eg, separating) the chains of the sequence that will be detected, then by reacting the chains with the two pairs of oligonucleotide probes in the presence of a heat-stable lobe. so that each pair of oligonucleotide probes is ligated together, then separating the reaction product and then cyclically repeating the procedure until the sequence has been amplified to the desired degree.The detection can then be carried out in a manner similar to that described. with respect to PCR.

B. Orynucleotide Probes As noted above, the methods of the present invention are useful for detecting DNA hybridization. The first step of the method includes counting between a DNA sample and an oligonucleotide probe to form a hybridized DNA. Oligonucleotide probes that are useful in the methods of the present invention can be any probe comprised between about 4 or 6 bases up to about 800 or 100 bases or more, most preferably between about 8 and about 15 bases. Oligonucleotide probes having any of a wide variety of base sequences can be prepared according to techniques that are well known in the art. Suitable bases for preparing the oligonucleotide probes can be selected from naturally occurring nudeotide bases such as adenine, cytosine, guanine, uracil and thymine; and non-naturally occurring or "synthetic" nucleotide bases such as 8-oxo-guanine, 6- mercaptoguanine, 4-acetylcytidine, 5- (carboxyhydroxyethyl) uridine, 2'-O-methylcytidine, 5-carboxymethylamino-methyl-2- thioridine, 5-carboxymethylaminomethyl-uridine, dihydrouridine, 2-O-methylpseudouridine, β-D-galactosylqueosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine , 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methyldtidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, β-D-mannosylkeosine, -methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N - ((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl) carbamoyl) threonine, N - ((9-β-D-ribofuranosyl) -purin-6-yl) N-methyl-carbamoyl) threonine, uridin-5-oxyacetic acid methyl ester, uridin-5-oxyacetic acid, wibutoxosin, pseudouridine, kerosine, 2-thiodothidine a, 5-methyl-2-thiouridine, 2-thiouridine, 2-thiouridine, 5-methylurdin, N - ((9-β-D-ribofuranosylpurin-6-yl) carbamoyl) threonine, 2'-O-methyl-5 -methyluridine, 2'-O-methylurdin, wferentsin and 3- (3-amino-3-carboxypropyl) uridine. Any oligonudeotide base structure can be employed, including DNA, RNA (although RNA is less preferred than DNA), modified sugars such as carbohydrates and sugars containing Z substitutions such as fluoro and methoxy. The oligonucleotides may be oligonucleotides in which at least one or all of the internudeotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholiths, phosphoropiperazidates and phosphoramidates (eg, any other phosphate residues). internucleotide bridging can be modified as described). The oligonucleotide can be a "peptide nudeic acid" such as that described in P. Nielsen et al., Science 254, 1497-1500 (1991). The only requirement is that the oligonucleotide probe must possess a sequence of at least a portion of which is capable of being bridged to a conodged portion of the sequence of the DNA sample. It may be desirable in some applications to count between the DNA sample and a number of oligonucleotide probes having different base sequences (e.g., when there are two or more objective nude acids in the sample, or when a target nucleic acid simple be hybridized to two or more probes in a "sandwich" test).

C. Hybridization Methodology The DNA (or nudeic acid) sample can be counted with the oligonucleotide probe in any suitable manner known to those skilled in the art. For example, the DNA sample can be solubilized in solution and brought into contact with the oligonucleotide probe by solubilizing the oligonucleotide probe in a solution with the DNA sample under conditions that allow hybridization. The suitable conditions are well known to those skilled in the art (see, e.g., patent of E.U.A. No. 4,358,535 to Falkow et al., And other references to US patents. that they have the same) and include conditions of high salt concentration. Alternatively, the DNA sample can be solubilized in solution, the oligonucleotide probe being immobilized on a solid support, whereby the DNA sample can be counted with the oligonucleotide probe by immersing the solid support having the DNA probe. oligonucleotide immobilized on it in the solution containing the DNA sample.

D. Oxidizing agents and oxidation-reduction reactions When a hybridization step precedes the oxidation step, after hybridization the hybridized DNA (or nucleic acid) is then brought to reaction with a suitable oxidizing agent that is capable of oxidizing a base preselected in the oligonucleotide probe in an oxidation-reduction reaction. The preselected base can be any nucleotide base that occurs naturally or synthetically in the oligonucleotide probe that is subjected to oxidation after reaction with the selected oxidizing agent. The preselected base exhibits a unique oxidation rate when paired compared to when the preselected base is unpaired. The preselected base must exhibit unique oxidation rates when paired with each of the four naturally occurring bases. Generally, bases can be detected whose 5'-mononucleotides (e.g., 5'-deoxyribonucleotide or 5'-ribonudeotide) exhibit rate constants per end of lO ^ M-ls ^ using the catalytic reaction. Examples of suitable preselected bases include but are not limited to guanine, adenine, 8-oxo-guanine, and 8-oxo-adenine, 8-bromo-guanine, guanosine, xanthosine, wiosin, pseudouridine, 6-mercaptoguanine, 8-mercaptoguanine, 2-thioxanthin, 6-thioxanthin, 6-mercaptopurine, 2-amino-6-carboxymethyl-mercaptopurine, 2-mercaptopurine, 6-methoxypurine, 2-acetylamino-6-hydroxypurine, 6-methylthio-2-hydroxypurine, 2-dimethylamino- 6-hydroxypurine, 2-hydroxypurine, 2-aminopurine, 6-amino-2-dimethylallyl-purine, 2-thioadenine, 8-hydroxyadenine and 8-methoxyadenine. Typically, the preselected base is selected from the group consisting of guanine, adenine, 6-mercaptoguanine, 8-oxo-guanine and 8-oxo-adenine, guanine being most preferred as a preselected base occurring naturally and 6-mercaptoguanine as the presently preferred synthetic pre-selected base. The oxidizing agent can be any molecule charged tai as a cationic, anionic or zwitterionic molecule that reacts with the preselected base at a single oxidizing potential. In this manner, the selection of the oxidizing agent will depend on the preselected base chosen in particular, and will be readily determined by those skilled in the art. Particularly preferred oxidizing agents include the transidon metal complexes which are capable of transferring DNA samples of metal with the preselected base so that the redudded form of the metal complex is regenerated, completing a catalytic cycle. Examples of transidon metal complexes suitable for use in the methods of the present invention include, for example, 2+ (2,2'-bipyridine) 3 ruthenium ("Ru (bpy) 32+"), + (4, 4, -dimethyl-2,2, -bipyridine) 3 ruthenium ("Ru (Me2-bpy) 32, 2+ (5,6-dimethyl-1,10-phenanthroline) 3 ruthenium (" Ru (Me2-phen ) 32 2+ (2I2, -bipyridine) 3 iron ("Fe (bpy) 32+"), 2+ (5-chlorophenanthroline) 3 iron ("Fe (5-CI-phen) 32+"), 2 + (2,2'-bipyridine) 3 of osmium ("Os (bpy) 32+ ,,)> 2+ (5-dorophenanthroline) 3 of osmium (" Os (5-CI-phen) 32+ "), 1 + dioxorenium phosphine and 1 + dioxorenium pyridine ("Re? 2 (py) 4 ^ +). Some anionic complexes useful as oxidizing agents are: Ru (bpy) ((S03) 2-bpy) 22- and Ru ( bpy) ((C02) 2-bpy) 2 ^ "some zwitterionic complexes useful as oxidizing agents are: Ru (bpy) 2 ((S? 3) 2-bpy) yu (bpy) 2 ((C? 2) 2- bpy. where (S? 3) 2 ~? y2- is 4,4'-sulfonate-2,2, -bipridine and (C? 2) 2-bpy ^ - is 4,4, -dicarboxy- 212'-bipyridine The appropriate substituted derivatives of pyridine, bipyridine and phenanthroline and their groups Pos can also be employed in complexes with any of the above metals. Suitable substituted derivatives include but are not limited to 4-aminopyridipa, 4-dimethylpyridine, 4-acetylpyridine, 4-nitropyridine, 4,4, -diamino-2,2'-bipyridine, 5,5'-diamino-2,2 '-bipyridine, β-diamino ^^' - bipyridine, 4,4'-diethylenediamine-2,2'-bipyridine, d.S'-diethylenediamine ^^ '- bipyridine, ß. ^ - diethylenediamine ^ .Z -bipyridine, 4,4, -dihydroxyl-2,2, -bipyridine, 5,5'-dihydroxyl-2,2'-bipyridine, 6,6'-dihydroxyl-2,2'-bipyridine, 4,4 ', 4" -triamine-2,2'2"-terpridine 4,4 \ 4" -trietilendiamin-2,2 ', 2"-terpyridine, 4,4,4,4" -trihydroxy-2,2,, 2"-terpyridine, 4,4,, 4" -trinitro-2,2,, 2"-tephyridine, 4,4,, 4" -triphenyl-2,2 ', 2"-ter? iridinium 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-dihydroxyl-1 , 10-phenanthroline, 3,8-dihydroxyl-1, 10-phenanthroline, 4,7-dinitro-1,10-phenanthroline, 3,8-dinitro-1,10-phenanthroline, 4,7-diferyl-1 , 10-phenanthroline, 3,8-diphenyl-1, 10-phenanthroline, 4,7-diis-eram-1, 10-phenanthroline, 3,8-disperamine-1,10-phenanthroline and dipyridop ^ -a ^ '^' - cfenfenzine. The oxidizing agent can be brought into reaction with the hybridized DNA according to any suitable technique, to carry out the oxidation-reduction reaction of the oxidizing agent with the preselected base. All that is required is that the oxidizing agent be brought into reaction with the DNA sample hybridized under suitably conditioned conditions to effect selective oxidation of the preselected base. For example, the transition metal can be reacted with solubilized hybridized DNA by solubilizing the oxidizing agent in the solution containing the hybridized DNA solubilized under sufficient conditions to allow the oxidation-reduction reaction to occur between the oxidizing agent and the preselected base. Alternatively, in the embodiment in which the hybridized DNA is immobilized on a solid support, the oxidizing agent can be brought into reaction with the hybridized DNA by immobilizing the oxidizing agent on the same solid support and immersing the solid support in a low solution. enough conditions to allow the oxidation-reduction reaction of the oxidizing agent and the preselected base to occur. The solvent in which the oxidation-reduction reaction takes place can be any suitable solvent to solubilize DNA; and preferably comprises water. Suitable conditions for allowing the oxidation-reduction reaction to occur will be known to those skilled in the art.

In a DNA or hybridized nucleic acid, the oxidizing agents are bound in the minor groove of the DNA, and in this way an intimate contact between the preselected base and the oxidizing agent is carried out by the unique structure of the double helix (or triple ). This protection of the residue from the preselected base creates the need for an electron tunnel through the solvent, which decreases the rate of electron transfer. The accessibility of the solvent varies with the nature of the nudeotide base that is paired with the preselected base. The tunnel distance can be calculated according to the formula: k / ks5 = exp (-ß? R) where \ r is the change in distance in the duplex compared to the single string, and ss is the velocity constant for the oxidization of the preselected base in the single-stranded DNA sample. In this way, the distance of the tunnel between the preselected base and the oxidizing agent is different for each base pairing and for each unpaired DNA. Therefore, the electron transfer rate constant indicates the identity of the paired (or unpaired) base. If the impulse force for the electron transfer is significantly less than the reorganization energy (?), A graph of RT in k against the driving force, corrected for work terms associated with the reactance range, produces a line with a tilt of 1/2, according to Marcus's theory. Based on Marcus's theory then, the absolute velocity constants can be calculated by the following equation: k = v exp [-ß (r-rrj)] exp [- (? G +?) 2/4? RT] in the that v is the velocity constant at the limit of controlled diffusion (101 1M "^ s_1), r is the distance between the reactive and the product in the adivate complex, rn is the distance of the closest approach of the readive and the product and ß is the influence of the intervening medium, because the preselected base, as noted above, is incorporated into the hybridized DNA, this imposes a finite distance through which the eledron must pass to the oxidizing agent. this way, r is not equal to ar. ß for the water is approximately 3A "1. This relatively large value for β indicates that significant changes in the electron transfer velocity constants will be carried out by very few changes in the distance of the tunnel. Since the conformation of the DNA between the preselected base and the paired base with the preselected base depends on the base paired with the preselected base, the base paired with the preselected base affects the distance of the tunnel through which the electron must pass between the preselected base and oxidizing agent. Therefore, a correlation is established between the distance of the tunnel and the specific base paired with the preselected base.

E. Detection of oxidation-reduction reactions The occurrence of the oxidation-reduction reaction may be arrested according to any suitable means conformed to the person skilled in the art. For example, the occurrence of the oxidation-reduction reaction can be stopped by using a detection electrode to observe a change in the electronic signal that is indicative of the occurrence of the oxidation-reduction reaction. Typically, a sensing electrode, responsive to electron transfer between the oxidizing agent and the hybrid DNA, is contacted with the solution containing the reactive hybrid DNA and the oxidizing agent. In addition to the detection electrode, a reference electrode and an auxiliary electrode are usually contacted with the solution (most of the current passes through the auxiliary electrode). Suitable detection devices are well known to those skilled in the art and include, for example, a crystalline carbon electrode or an indium tin oxide electrode. Similarly, suitable reference electrodes are known in the art and include, for example, silver / silver durometer alloys. The detection of the electronic signal associated with the oxidation-reduction reaction allows the determination of the presence or absence of hybrid DNA. The step of determining the presence or absence of hybrid DNA typically induces i) measuring the reaction velocity of the oxidation-reduction, ii) compare the measured reaction rate with the oxidation-reduction reaction rate of the transition metal complex with a single-stranded DNA, and then iii) determine whether the measured reaction velocity is essentially the same as the velocity of oxidation-reduction reaction of the transidon metal complex with single-stranded DNA. The step of measuring the rate of reaction can be carried out by any suitable means. For example, the relative reaction velocity can be determined by comparing the current as a function of scanning rate, probe concentration, target concentration, mediator, pH regulator, temperature, and / or erochemical method. The oxidation-reduction reaction rate can be measured according to the suitable means conformed by the person skilled in the art. Typically, the oxidation-reduction reaction rate is measured by determining the eledronic signal associated with the occurrence of the oxidation-reduction reaction. For example, the electronic signal associated with the oxidation-reduction reaction can be measured by providing a suitable apparatus in electronic communication with the detection electrode. A suitable apparatus will be able to measure the eledronic signal that is generated, in such a way as to provide a measurement of the oxidation-reduction reaction rate of the reaction of the hybrid DNA and the oxidizing agent. The electronic signal can be caraderistic of any electrochemical method, including cyclic voltammetry; normal pulse voltammetry, cronaamperometry, and square wave voltammetry, cyclic voltammetry being the currently preferred form. The measured reaction rate can then be compared to the known oxidation-reduction reaction rate of the transition metal complex with a single-stranded DNA. As discussed in detail above, the tunnel distance between the oxidizing agent and the selected base in the DNA, either hybrid or single chain, affects the rate of oxidation-reduction reaction between the oxidizing agent and the preselected base. Therefore, the hybrid DNA exhibits a different oxidation-reduction reaction rate than that of the single-stranded DNA. The presence or absence of hybrid DNA in the preselected base can be determined by determining whether or not the measured oxide-reduction reaction velocity is the same as the oxidation-reduction reaction rate of the oxidizing agent and the preselected base in the DNA of a single chain. Furthermore, the tunnel distance between the oxidizing agent and the preselected base differs according to the distance of the ligation between the preselected base and its pair, so that each possible base pairing can be distinguished from the others. The bonding distance between the preselected base and its base pair depends on the base that is paired with the preselected base. For example, the oxidation-reduction reaction rate for guanine oxidized paired with adenine differs from the oxidation-reduction reaction velocity for the guanine oxidized paired with cytosine, which in turn is different from the reaction rate of Oxidation-reduction for the guanine-paired guanine oxidation, which is also different from the oxidation-reduction reaction rate for the oxidation of guanine paired with thymine. More specifically, the oxidation-reduction reaction rates for guanine oxidation follow the trend in which the guanine of a single chain is greater than that of guanine paired with adenine, which is greater than that of guanine paired with guanine, which is greater than that of guanine paired with thymine, which is greater than that of guanine paired with cytosine. Therefore, the methods of the present invention are useful for detecting mismatches of a single base pair in the preselected base or base pair adjacent to the preselected base. Advantageously, the distinction between the oxidation-reduction reaction rates of the preselected base oxidation when pairing with each of the different bases of natural occurrence, also allows identification of the base paired with the preselected base. The base paired with the preselected base can be identified i) by measuring the oxidation-reduction reaction rate detected, ii) by comparing the measured reaction rate with each of the four different known oxidation-reduction reaction rates of the oxidizing agent with a DNA having adenine, cytosine, guanine, or thymine attached to the preselected base, and iii) determining which of the known oxidation-reduction reaction rates is essentially the same as the measured reaction rate. The reaction velocity can be measured according to the techniques described above. Similarly, the reaction rates of each of the four different oxidation-reduction reactions of the oxidizing agent with a DNA having adenine, cytosine, guanine or thymine attached to the preselected base can be measured in accordance with the same techniques. that these velocities of reacdón be known. The measured reaction velocity of the oxidation-reduction reaction of the oxidizing agent with the hybrid DNA can then be compared with the known oxidation-reduction reaction rates of the oxidizing agent with DNA having adenine, dtosin, guanine or thymine attached to the base preselected For example, the base paired with the preselected base is determined by determining the coupling of the solid base having the oxidation-reduction velocity, which is essentially equal to the measured oxidation-reduction reaction rate.

F. Determination of the DNA sequence The present invention also provides a method of Determination of the DNA sequence comprising a) counting a DNA sample with an oligonucleotide probe to form a hybrid DNA; the oligonucleotide probe induces a preselected synthetic base having a unique oxidation potential; b) reacting the hybrid DNA with a tai oxidizing agent such as a transidon metal complex, capable of oxidizing the preselected synthetic base in the nucleotide probe in an oxidon-reduction reaction, the oligonucleotide probe has a predetermined number of pre-selected synthetic bases; c) stop the oxidation-reduction reaction; d) measuring the speed of the oxidation-reduction reaction detected; and e) identify the base paired with the preselected synthetic base. As in the methods discussed hereinabove, the DNA sample can be amplified before the spot passage with the oligonucleotide probe, according to techniques known to the person skilled in the art. The synthetic base can be selected from the group of bases described hereinabove and other synthetic bases known to the person skilled in the art. The only limitation is that the synthetic base must possess a unique oxidizing potential compared to the oxidized potendals of the four bases that occur in nature, namely, adenine, cytosine, guanine and thymine. The steps of contacting the DNA sample with the oligonucleotide probe; the reaction of the hybrid DNA with the oxidizing agent, the detection of the oxidation-reduction reaction, and the measurement of the reaction rate, can be carried out as described hereinabove. The step of identifying the paired base with the preselected synthetic base involves the steps of (i) comparing the measured reaction rate for each of the four different oxidizing-reducing reaction velocities condescending the oxidizing agent to the DNA having adenine , dtosine, guanine or thymine linked to the preselected synthetic base; (ii) determining which of the known oxidation-reduction ratios is essentially the same as the measured reaction velocity. In another embodiment, the oligonucleotide probe also includes a preselected second synthetic base. The preselected second synthetic base has a unique oxidation potential that is different from the oxidation potential of the preselected synthetic first base. In this embodiment, the step of detecting the oxidation-reduction reaction of the oxidizing agent with the preselected base also induces detection of the oxidation-reduction reaction of the oxidizing agent also with the preselected second synthetic base. In addition, the measurement step of the oxidation-reduction reaction velocity also includes measuring the oxidation-reduction rate of the oxidation of the preselected second base by means of the oxidizing agent. In addition, the identification step of the paired base with the preselected synthetic base also includes identifying the paired base also with the second preselected synthetic base. According to this embodiment, the oxidation-reduction reactions of preselected bases can be detected so that finally the bases that are paired with each preselected synthetic base can be identified using the method described hereinabove. As will be apparent to the person skilled in the art, the above method can be carried out with more than two pre-selected synthetic bases, provided that each preselected synthetic base exhibits a unique oxidation potential different from the oxidation potential of all other preselected synthetic bases, and different from the oxidation potential of each of the four bases that occur in nature. Since each base that pairs with a preselected base can be identified according to the methods described in this, DNA sequencing can be determined by repeating the steps of the above method with a suffcient number of probes different from the oligonucleotide having the preselected synthetic base at different sites to identify each base in the DNA sample. In other words, the DNA sequence of the sample can be determined by providing a sufficient number of oligonucleotide probes in which each probe sequence induces at least one of the preselected synthetic bases, and the synthetic base is located at a different site and calculated along the sequence of the probe in each oligonucleotide probe. In this way, repeated detection of the oxidation-reduction reaction of the hybrid DNA with an oxidizing agent, the measurement of the oxidation-reduction rate of the reaction, and the identification of the base paired with the preselected synthetic base will result in the base identification by base of the sequence of the DNA sample.

G. Apparatus The present invention also provides an apparatus useful for carrying out the methods of the present invention. One of these illustrative apparatuses is shown schematically in Figure 3. In general, the apparatus comprises a plurality of DNA sample containers 10. A driving assembly 11 serves as a means of handling samples to carry the plurality of DNA sample containers. A liquid reservoir 12, a feeder line 13 and a valve 14 serve as an oligonucleotide probe release means for releasing the oligonucleotide probe in each of the DNA sample containers, and a corresponding reservoir of liquid 15, line of feed 16 and valve 17 serve as a means of releasing oxidizing agent to release the transidon metal complex to each of the plurality of DNA sample containers. A probe assembly 20 including a condudor 21 and a probe 22 serves as an oxidizing-reduction reaction medium for detecting an oxidation-reduction reaction. In operation, the DNA samples are pre-deposited in the sample containers 10. The conduit assembly 11 then consecutively transports the sample containers 10 below the oligonucleotide probe delivery means and the oxidizing agent release means to release the samples. reactive respects in them. After the release of the readive, the respective sample container advances by means of the conduction means to a position below the probe 22, and the probe 22 advances by means of the conductor 21 and towards the sample container for detection of the Oxidation-reduction reaction. With the probe 22 are carried adidonales eledrodos, necessary to carry out the cyclic voltamogram. The operation of the different components and the data collection can be carried out with an appropriate controller 30, such as a software program running on a suitable computer for general use. Of course, numerous variations on the above apparatus will be readily apparent to one skilled in the art. The plurality of DNA sample containers can be of any suitable container known to the person skilled in the art, and includes microtiter plates, test tubes, Petri dishes, culture bottles, solid supports and the like, which are capable of containing the DNA sample. The sample handling means can be any means of handling properly designed sample containers, known to those skilled in the art, which are capable of carrying the DNA sample containers. Oligonucleotide probe delivery means suitable for releasing the oligonucleotide probe to each of the DNA sample containers are well known in the art. For example, according to one embodiment, the oligonucleotide probe delivery means comprises a solid support on which the oligonucleotide probe is immobilized. The oligonucleotide probe release means should allow sufficient counting between the DNA sample and the oligonucleotide probe under appropriate conditions to effect hybridization of the DNA sample and the oligonucleotide probe. Suitable oxidizing agent releasing means for releasing the oxidizing agent to each of the plurality of DNA sample containers are well known in the art. For example, according to one embodiment, the oxidizing agent is fixed to a solid support comprising the oxidizing agent release medium. The oxidation-reduction reaction detector for detecting an oxidation-reduction reaction, according to one embodiment, may comprise one or more electrodes that are capable of detecting the oxidation of the preselected base. Suitable reference detectors and eyelid electrodes were described hereinabove with reference to the methods of the present invention. Preferentially, the eledrodes are in electronic communication with a means to measure the oxidation-reduction reaction velocity. Suitable means for measuring the oxidation-reduction reaction velocity are known to the person skilled in the art, as described hereinabove. In an alternative embodiment of the apparatus of the present invention, the apparatus for detecting DNA hybridization comprises (a) a DNA sample container; (b) an oligonucleotide probe delivery means for releasing a plurality of oligonucleotide probes to the DNA sample container; (c) a means of releasing oxidizing agent to release the oxidizing agent to the DNA sample container; (d) an oxidation-reduction reaction detector for detecting an oxidation-reduction reaction; this apparatus is adapted for use with immobilized probes such as those described in U.S. Patent Nos. 5,143,854 and 5,405,783 to Pirrung et al .; Fondor, et al., Nature 364: 555 (1993); Bains, Agnew. Chem. 107: 356 (1995); and Noble, Analitical Chemistry 67 (5): 21 (1995), the descriptions of which are incorporated herein by reference in their entirety. As indicated above, the DNA sample container can be any suitable container suitable for the person skilled in the art. The oligonucleotide probe cleavage medium is preferably a solid support having a plurality of oligonucleotide probes immobilized thereon, which is capable of delivering the probes to the DNA sample container. For example, in accordance with one embodiment, the solid support having the plurality of oligonucleotide probes immobilized thereon, is put in place with the DNA sample within the DNA sample container under suffi cient conditions to allow hybridization of the DNA sample with one or more oligonucleotide probes. The oxidizing agent releasing means suitable for releasing the oxidizing agent to the DNA sample container were described hereinabove. Preferred oxidizing agent release means comprises a solid support having the oxidizing agent immobilized thereon. In accordance with a preferred embodiment, the oxidizing agent and the plurality of oligonucleotide probes are immobilized on the same solid support. The apparatus according to the present invention is useful for testing diagnostic tests of a variety of DNA samples. The plurality of oligonucleotide probes allows the testing and detection of a variety of DNAs within a single sample, thus providing a useful tool for the selection of a single sample from a variety of DNAs, including pathogens, viruses, and the like.

H. Detection of RNA hybridization. RNA sequencing. v detection of RNA mismatch Also described herein are RNA hybridization detection methods, RNA sequencing methods and RNA mismatch detection methods. Et RNA useful in carrying out these methods includes, but is not limited to, ribosomal RNA, transfer RNA or genomic RNA (e.g., RNA obtained from RNA viruses such as retroviruses, HIV-1, etc.). A first aspect of the present invention is, therefore, a method of detecting RNA hybridization comprising: (a) counting a sample of RNA with an oligonucleotide probe to form a hybrid RNA; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing a preselected base in the oligonucleotide probe in an oxidoon-reduced reaction, the oligonucleotide probe having at least one of the preselected bases; (c) detecting the oxidation-reduction reaction; (d) determining the presence or absence of hybrid RNA of the oxidation-reduction reaction detected in the preset base. More particularly, an RNA hybridization detection method comprises: (a) contacting an RNA sample with an oligonucleotide probe to form a hybrid RNA; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing a preselected base in the otigonucleotide probe in an oxidoon-reduced reaction, the oligonucleotide probe having at least one of the preselected bases; (c) detecting the oxidoon-reduced reacton; (d) measuring the reaction rate of the oxidation-reduction detected; (e) comparing the measured reaction rate with the oxidation-reduction reaction velocity of the transidon metal complex with a single-stranded RNA; and then (f) determining whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the transidon metal complex with single-stranded RNA. An RNA sequencing method comprises: (a) counting an RNA sample with an oligonucleotide probe to form a hybrid RNA, the oligonucleotide probe includes a preselected base having a unique oxidation rate; (b) reacting the hybrid RNA with a transidon metal complex capable of oxidizing the preselected base in the oligonucleotide probe in an oxidation-reduction reaction, the oligonudeotide probe has a predetermined number of preselected bases; (c) detecting the oxidation-reduction reaction; (d) measuring the reaction speed of the oxidized-reduction reactant; and (e) identify the base paired with the preselected base. Oligonucleotide probes, hybridization methodology, oxidation agents, detection of oxidation-reduction reactions, and apparatuses useful for carrying out these methods are essentially as indicated in the sections AH above, adapted for use with RNA as the acid sample. nucleic acid, in accordance with the principles known to the person skilled in the art (for example, uradro replaces thymine as a base).

I. Detection of the preselected base on the target nucleic acid In the methods specifically described above, the metal complexes are used to obtain an electrochemical current from DNA or single-stranded or double-stranded nucleic acids. Preselected bases such as guanine give an echerochemical signal, and this signal is much weaker for double-stranded DNA. These methods advantageously exhibit a high strudural sensitivity, and can resolve a single base mismatch. Therefore, these methods are particularly advantageous for DNA sequencing. However, two disadvantages of these methods are that: (a) there is a negative signal that runs from the chain of the probe to the hybrid, and (b) there is no amplification of the signal. The following techniques provide solutions to this problem. further, the following techniques are particularly useful for diagnostic tests, and are particularly useful for the quantitative detection of nucleic acids. In view of the foregoing, a method of detecting the presence or absence of a target nucleic acid in a test sample suspected of containing it, in which the target nucleic acid contains at least one of the present invention, is also described herein. a preselected base. In contrast to the methods described above, in the present method the preselected base is located on the target nudeic acid, instead of being on the oligonudeotide probe. The method can be carried out in a test sample containing the target nudeic acid. Any test sample suspected of containing the target nucleic acid may be used, including (but not limited to) tissue samples such as biopsy samples and biological fluids such as blood, sputum, urine and semen samples, Baderian cultures, samples of soil, food samples, etc. The target nucleic acid may be of any origin, including animal, plant or microbial (eg, viral, prokaryotic and eukaryotic baderian, protozoan, fungal, protist, etc.), depending on the particular purpose of the test. The sample may be treated or purified before carrying out the present method in accordance with techniques known or apparent to the person skilled in the art.; and if so desired, the nucleic acids herein can be digested, fragmented, and / or amplified (see above) before carrying out the present method. As illustrated schematically in Figure 4, the method comprises (a) counting the test sample with an oligonucleotide probe that specifically binds to the target nucleic acid to form a hybrid nucleic acid; (b) counting the hybrid nucleic acid with a transidon metal complex that oxidizes the preselected base in an oxidation-reduction reaction; (c) detecting the presence or absence of the oxidation-reduction reaction associated with the hybrid nudeic acid; and (d) determining the presence or absence of the target nucleic acid in the test sample of the oxidation-reduction reaction detected in the preselected base. As illustrated in Figure 4, the oligonucleotide probe can be immobilized on a solid support to facilitate separation of the test sample from the hybrid nucleic acid, with the separation step occurring before the detection step (e.g., between steps (a) and (b) or between steps (b) and (c)). Alternatively, the oligonucleotide probe can be supplied free in solution, and other means can be provided for separating the hybrid nucleic acid from the sample (for example by a nude mediator bond that binds to the oligonucleotide probe, or by a biotin binding interaction). -avidin, in which biotin binds to the probe of the oligonucleotide and avidin is immobilized on a solid support). Preferably, the target nucleic acid contains at least ten more of the preselected base than the oligonucleotide probe, or most preferably at least 50 or 100 more of the preselected base than the oligonucleotide probe. A greater current increase is advantageously obtained when the objective nudeic core contains more of the preselected base than the oligonucleotide probe. Oponally, but preferably, the oligonucleotide probe is free from the preselected base, or is at least essentially free from the preselected base (it is suffi- ciently less than the preselected base so that the signal from the probe does not interfere with it, neither be taken by mistake as a signal of the target nucleic acid). Where a naturally occurring base sequence is not available that hybridizes conveniently with the target nude, the strategy of employing alternative bases that are inadivas for redox can be used (discussed below). The target nucleic acid is preferably larger than the oligonucleotide probe, and at least one of the preselected bases does not hybridize with the oligonucleotide probe in the hybrid nucleic acid (ie, it is a "hanging" base), as illustrated in Figure 4. Preferably, at least 10, 50 or 100 of the preselected bases are "hanging" bases, thereby providing substantial amplification of the detected electrochemical signal. For example, an oligonucleotide probe that does not contain any guanine residue (eg, only A, T, and C) can be used. The cyclic voltamogram of Ru (bpy) 32+ in the presence of this chain is very similar to that which does not have the oligomer. This chain is then hybridized with a guanine-containing target chain, either in the overlapping paired bases regions or in the hanging regions, or both (as illustrated in Figure 4 by means of a "G" adjacent to the target nucleic acid chain), if the target nudeic acid is larger than the oligonucleotide probe. Because multiple guanines are detected, the signal is amplified in relation to the number of hybrids formed. In the case in which the target chain is genomic DNA or RNA, large numbers of hanging guanines are found, which would give a huge signal amplification. For example, ribosomal RNA can contain up to 1, 000 guanines for a particular organism, and therefore could provide approximately an amplification of 1,000 times per hybridization event. For example, in a preferred embodiment, the test for the preselected base on the target chain includes immobilization of the probe chain (preferably inactive to redox) on a solid surface oriented near the surface of the electrode, which provides a signal low base when it is scrutinized in the presence of the mediator. The solid surface is then counted with a solution of the target chain, which contains the preselected base. If hybridity occurs, the target chain will now be in close proximity to the electrode, and an increase in current will be stopped, Quantification of nucleic acids. The present method is particularly well suited for the quantitative detection of nucleic acids. In the cases described in this section, the rate constant for oxidation of the hybrid by means of the oxidizing agent (e.g., Ru (bpy) 33+), can be determined from the cyclic voltammogram (or other electronic signal) by digital simulation. Under most conditions, this reaction follows the second-order dnetics, so that the velocity = k [Ru (bpy) 32 +] [DNA], where k is ta constant velocity that is specific to the particular hybrid probe-target, [Ru (bpy) 32+] is the concentrator of the oxidizing agent, and [DNA] is the concentration of the hybrid (which could be a DNA-RNA hybrid). If k and [Ru (bpy) 32+ are known), then the amount of the hybrid can be determined. In practice, a calibration curve is constructed for current increments obtained with different amounts of standard soludons containing target DNA or RNA, and the current increase is used to obtain the amount of hybrid directly. This amount is then directly related to the amount of target material (eg, infectious organisms in a clinical sample). See for example, M. Hotodniy et al., J. Virol. 69, 3510-3516 (1995); J. Mellors et al., Sdence 272, 116-1170 (1996). The oligonucleotide probes, the hybridization methodology, the oxidizing agents and the oxidoon-reduction reaction methods, the detection of oxidizing-reduction reactions, and the devices useful for carrying out these methods are as indicated in sections AH previous J. Alternative Bases Which Are Inactive for Oxidation-Reduction A disadvantage of the method described in section H above is that the oligonudeotide probe preferably does not contain a substantial number of the preselected base (eg, guanine). One solution to this problem is to use an alternative base that could replace guanine (ie, a base that, like guanine, has a higher binding affinity for atocin than the other bases in a double nudeic acid chain) in the Probe chain, but not oxidized by the oxidizing agent under the applicable reaction conditions. Examples of these alternative bases when guanine is the preselected base are inosine and 7-deazaguanine. In this manner, a method of detecting a target nucleic acid wherein the nucleic acid contains at least one preselected base and the probe or nucleic acid capture contains inactive alternative bases to the oxidoreduction comprises: a) contacting the nudeic acid target with a complementary nudeic acid that specifically binds to the target nucleic acid to form a hybrid nucleic acid; b) reacting the hybrid nucleic acid with a transidon metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; c) stop the oxidoon-reduced reaction; and d) determining the presence or absence of nucleic acid of the oxidation-reduction reaction detected in the preselected base. When the preselected base in the target nucleic acid is guanine and the target nucleic acid contains dtodna (which ordinarily would bind with guanine in the complementary nucleic acid), then the complementary nucleic acid contains an alternative base that binds to the dtodna in hybrid nude acid. The alternative base can be selected from the group consisting of inosine and 7-deazaguanine. The reaction step typically comprises the reaction of the transidon metal complex with the nucleic acid under conditions sufficient to effect selective oxidization of the preselected base without oxidizing the alternative base. The oligonucleotide probes, the hybridization methodology, the oxidizing agents, the oxidation-reduction reaction methods, the detection of oxide-reduction reactions and the devices useful for carrying out these methods, are as indicated in the previous sections .

K. Polymerization of the preselected base with terminal transferase An alternative embodiment of the method described in section H above includes elongating the target nudeic acid with terminal transferase to provide additional bases thereto to those pre-selected. As illustrated in Figure 7, this method comprises: a) contacting the test sample with an oligonucleotide probe that specifically binds to the target nucleic acid to form a hybrid nudeic acid, the oligonucleotide probe has terminal ends that are blocked for elongation by terminal transferase; b) contacting the oligonucleotide probes with a solution containing a preselected base in the presence of terminal transferase to produce an extension product of the target nucleic acid, the extension product comprising the preselected base; c) counting the oligonucleotide probe with a transition metal complex that oxidizes the preselected base in an oxidon-reduced reaction; d) detecting the presence or absence of the oxidation-reduction reaction; and e) determining the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. The test samples are preferably separated from the oligonucleotide probe before the detection step, and are most preferably separated from the probe between steps a) and b) above. Separation can be accomplished by the use of an immobilized probe, or the probe can be provided free in solution, as discussed in section H above.

The oligonucleotide probes, the hybridization methodology, the oxidation-reduction methods and the oxidizing agents, the detection of the oxidation-reduction reactions and the devices useful for carrying out these methods are as indicated in Previous sections.

L. Sandwich tests A further embodiment of the method in section H above is the so-called "sandwich" test, illustrated schematically in Figure 8. In a sandwich test, the target nucleic acid is part of a hybrid of three (or more) ) members, comprised of a capture probe, the target nudeic acid and the signal probe. A method of detecting the presence or absence of a target nucleic acid in a test sample that is suspected to contain the same comprises: a) providing an oligonucleotide capture probe, wherein the capture probe specifically binds to the acid target nucleic acid; b) contacting the test sample with the capture probe to form a hybrid nucleic acid; c) counting an oligonucleotide signal probe with hybrid nude acid, wherein the signal probe specifically binds to the target nucleic acid, and wherein the signal probe contains at least one preselected base, to produce a hybrid nucleic acid sandwich; d) contacting the hybrid nucleic acid sandwich with a transition metal complex that oxidizes the preselected base in an oxidon-reduced reaction; e) detecting the presence or absence of the oxidation-reduction reaction associated with hybrid nudeic acid; and f) determining the presence or absence of target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. Preferably, the test sample is separated from the capture probe; this separation step may occur between step b) and step c) above, or between step c) and step d) above. Depending on the format of the test (eg, heterogeneous or homogeneous), the oligonucleotide capture probe may be immobilized on a solid support (eg, a polymeric bead, a plate or the inner surface of a well of microtiter plate) , or alternative means provided to separate the hybrid nucleic acid from the test sample, as discussed above. Numerous "sandwich" test formats are known. The choice of the test format is not critical, and any suitable format can be used to carry out the present invention. For example, the oligonucleotide capture probe may be immobilized on a solid support, as described in U.S. Pat. No. 4,486,539 for Ranki et al. The oligonucleotide probes may contain a polymer forming unit, as described in US Pat. No. 4,868,104 to Kurn et al., And the hybrid nucleic acid sandwich can be separated therefrom by polymerization. The signal probe can be linear or branched, as described in M.S. Urdea, Clinical Chem. 39, 725-726 (1993). A mediator polynucleotide that binds to the oligonucleotide capture probe for an immobilized polynucleotide, as described in US Pat. No. 4,751,177 for Stabinsky. The oligonucleotide probe can be attached to a member of a specific binding pair (eg, biotin), and the hybrid nucleic acid sandwich can be separated from the test sample by means of a second binding interaction with the other member of the nucleic acid sandwich. binding pair, which is immobilized on a solid support (e.g., avidin), as described in R. Goodson, EPO Application 0 238 332: W. Harrison, EPO Application 0 139 489, and N, Dattagupta, Solidtud EPO 0 192 168. Oligonucleotide probes, hybridization methodology, oxidizing agents and oxidation-reduction reaction methods, the detection of oxidizing-reduction reactions, and the apparatus used to carry out these methods are they give as in the previous AK sections, M. Detection of the preselected base in the presence of the background guanine signal The presence of a preselected base in an oligonucleotide probe can be arrested even in the presence of a background signal produced from the guanine oxidation. Because the non-mating detec- tion depends on the ability to detect a preselected base in the oligonucleotide probe in the presence of the four native bases (A, T / U, C, and G), therefore, the preselected base must be able to be oxidized more quickly than the other four bases. The present invention provides an oligonucleotide probe useful for the eledrochemical detection of a preselected base in the presence of the background guanine signal. The oligonucleotide probe can consist of any oligonucleotide probe as given in section B above, wherein at least one purine base in the oligonucleotide probe is a purine substituent of formula I: The oligonucleotide probe may contain as many bases of the above formula as desired (eg, 1, 2 or 3, up to 5, 10, 15 or more), depending on the desired binding complement thereof. Specific examples of said oligonudeotide probes, and nudeotides useful for the preparation thereof, are the compounds of formula II: wherein: R1 is HO-P (O) (OH) -O-, a nucleotide, or an oligonucleotide; R2 is -H, a nucleotide or an oligonucleotide; R3 is -H, -OH, halogen (eg, fluorine, doro), alkoxy (eg, C1-C4 alkoxy, such as methoxy or ethoxy), amino, or azido; and R4 is -O- or -CH2-. The oligonucleotide probes described in relation to the above formulas I and II are made in accordance with known techniques, modified in the light of the examples described below, as will be readily apparent to those skilled in the art. In a preferred embodiment of the compound of formula II, R- is HO-P (O) (OH) -O-. In another preferred embodiment of the compound of the formula I, R is -H. When R- | is a nudeotide or an oligonucleotide, the phosphodiester ligation is for the 3 'terminal end. When R2 is a nucleotide or oligonucleotide, the phosphodiester ligation is for the 5 'terminal end. The compounds of the formula I are advantageously included as a base in an oligonucleotide probe which can be used in the methods of the present invention, as described in the above A-M sections. The oligonucleotide probe can, in fact, include multiple bases, but must include at least one base of the formula I when the oligonucleotide probe is to be used for the detection of a preselected base in the presence of the background guanine. The oligonucleotide probe may be 5, 10, 50 or up to 100 base pairs in length. A particular example of a compound of formula II is 6-mercaptoguanosine 5'-monophosphate (6-S-GMP).

N. Electrode Structures An electrode useful for the electrochemical detection of a preselected base in a nucleic acid in accordance with the methods described above comprises: (a) a conduding substrate having an active surface formed thereon; and (b) a polymer layer connected to the adipose surface. The polymer layer is one that binds to the nucleic acid (eg, by hydrophobic interaction or any other suitable binding technique), and is porous to the transition metal complex (i.e., the transition metal complex can migrate fairy the nucleic acid bound to the polymer). The conductive substrate can be a metallic substrate or a non-metallic substrate, including semiconductor substrates (e.g., gold, glassy carbon, tin oxide doped with indium, etc.). The conductive substrate can take any physical form, such as an elongate probe having an adipose surface formed on one end thereof, or a thin sheet having the active surface formed on one side thereof. The polymer layer can be connected to the active surface by any suitable means, such as by clamping the polymer layer to the adipose surface, evaporating a solution of the polymer onto the electrode, or electropolymerizing. Exemplary polymers include, but are not limited to, nylon, nitrocellulose, polystyrene and poly (vinylpyridine). The thickness of the polymer layer is not critical, but it can be 100 Angstroms (A) up to 1, 10 or even 100 microns. The method can be used essentially all the methods described in the previous A-M sections. Thus, in general, the present invention provides a method for detecting a nudeic acid, said nucleic acid containing at least one preselected base, the method comprising: (a) contacting a sample containing said nudeic acid with an electrode, electrode comprising a conductive substrate having an adiva surface formed therefrom and a polymer layer as described above in relation to the active surface; (b) making the nudeic acid reacted with a transidon metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; (c) stopping said oxidation-reduction reaction by measuring the current flow through said electrode; and (d) determining the presence or absence of the nudeic acid from the detected oxidation-reduction reaction in the preselected base.

O. Microelectronic devices One advantage of the techniques described above is that they can be carried out with a microelectronic device. A microelectronic device useful for the electrochemical detection of a nucleic acid species in the methods described above comprises a microelectronic substrate having first and second opposing surfaces; a conducting electrode in the first surface; and an oligonucleotide capture probe immobilized on the first surface adjacent to the conductive electrode. The capture probe is suffi- ciently separated near the adjacent electrode (eg, from about 0.1, 1 or 2 μ, to about 50, 100, 500 or even 1000 μ), so that an oxidation-reduction reaction occurring in that probe, or in a target nucleic acid hybridized with that probe, is detected in the adjacent electrode. In the preferred embodiment shown in FIGS. 9 and 10, a microelevated device 20 has a plurality of electrodes spaced 21 apart on the first opposing surface, and a plurality of separate oligonucleotide capture probes 22 immobilized adjacent to each of the separated electrodes. By supplying a plurality of separate oligonudeotide probes, differing from each other, each with an associated module, a single compact device is provided which can stop several different hybridization events. Each electrode is connected elhedrally to a suitable contact 23, so that the device can be connected or otherwise operatively housed with the electronic equipment necessary to carry out the steps of detection and determination of the methods described in the present invention. The nucleic acid can be immobilized sequentially at the appropriate site on the microelectronic substrate by known techniques. See, for example, Patent of U.S.A. No. 5,405,783 by Pirrung and others. The microelectronic substrate may be a semiconductor (for example, silicon), or non-semiconductor materials that may be processed using convective microelevated techniques (e.g., glass). The electrode can be metal or a non-metallic conductive material, such as polycrystalline silicon. The eledrode can be formed using conventional microelectronic processing techniques, such as acid etching by deposition. Various microeledronic stringency and suitable manufacturing techniques are well known to those skilled in the art. See, for example, S.M. Sze, VLSI Technology (1983); S.K. Ghandhi, VLSI Fabrication Principies (1983). The following examples are provided to describe the present invention, and should not be considered as limiting thereof. In these examples, crr ^ / s means square centimeters per second. M stands for molar concentrate, M "^ s" ^ means moles per second, eV stands for eledrovolts, V stands for volts, nm stands for nanometers, GMP stands for guanosine 5'-monophosphate, and ITO stands for tin oxide impurified indium electrode. Cyclic voltammograms were assembled with an EG + G Model 273A potendostat / galvanostat from Princeton Applied Research, in accordance with known techniques. The ITO work wheels are manufactured from a glass sheet of soda-lime coated with ITO, part number CH-50IN-1514, available from Delta Technologies, Ltd, 13960 North 47 th Street, Stillwater, Minnesota 55082 -1234 US Nylon film is available as HYBOND-N + nylon membrane, catalog number RPN 1210B, from Amersham Corp, 2636 Ctearbrook Drive, Ariington Heights, IL 60005 EU A.

EXAMPLE 1 Measurement of the Rui cyclic voltammogram bpy) ^ 2 + The cyclic voltammograms of Ru (bpy) 32+, with and without calf thymus DNA, are shown in Figure 1, with the catalytic increase produced by the multiple rotations of oxidation of DNA by the oxidized form of the metal complex observed during an individual electronic voltammetric scan. The voltammetry of any redox pair bound to DNA must be analyzed in terms of a quadratic scheme that relates the bound and unbound forms because the diffusion coefficient of the DNA is much smaller (ie, 2.0 x 10-7 crr ^ / s ) than that of the metal complex (8.0 x 10"6 cm2 / s) This phenomenon generally leads to dramatically diminished currents for the linked form; however, at high enough ionic strength [Na +] = 0.8 M), the binding of the metal complex is too weak to affect the response to the current. In this case, the current can be analyzed in terms of a simple EC mechanism.

Ru (bpy) 32+? Ru (bpy) 33+ (E) Ru (bpy) 33+ + DNA? Ru (bpy) 32+ + ADNox (C *) EXAMPLE 3 Analysis of cyclic voltamorams Cyclic voltammograms were analyzed by adjusting the complete current potential curves, subtracting the background, using the DIGISIMMR data analysis package. The input parameters were E-j / 2 for the metal complex and the diffusion coef fi cients for the metal complex and DNA, all of which were determined in separate experiments. Therefore, the only parameter obtained from the adjustment was the second order rate constant for equation 2, k = 9.0 x 103M_1s-1. This same velocity constant was determined on a wide scale of scrutiny velocities: The rate constant for DNA oxidation by Ru (bpy) 33+ was confirmed in two separate experiments. First, square wave voltammograms were used to obtain a first order pseudo kobs for equator 2 by adjustment with the COOLMR algorithm. The COOL ^ R algorithm uses an adjustment approach that is significantly different from DIGISIM ^ R; however, the kODS versus DNA plots were linear and gave a second order velocity constant of k = 8.2 x 103 M "1s-1, which matches the velocity constant obtained from the adjustment of cyclic voltammograms with DIGISIM ^ A. Secondly, authentic samples of Ru (bpy) 33+ were prepared and reacted with DNA directly in a blocked fast-paced flow The overall analysis of the time-dependent spectra between 350 and 600 nm showed that Ru (bpy) ) 33+ became cleanly Ru (bpy) 3-2 without any intermediary and at a velocity constant of 12 x 103 M "1s-1. Thus, the velocity constant for the DNA oxidation by Ru (bpy) 33+ was firmly established by two independent electrochemical measures with dramatically different adjustment protocols and by a non-electrochemical, blocked flow technique with adjustment of the entire visible stapes.

EXAMPLE 3 Analysis of cyclic voltammograms If the directing force for electron transfer is significantly less than the reorganizational energy (\), a graph of the In k of RT against the driving force (when corrected for useful terms associated with the focus of the readivos) should give a line reda with a slope of 1/2. The rate constants for the oxidation of DNA by several Metat (bpy) 33+ derivatives with different redox potentials are shown in Table 1 below. Since Marcus's theory describes the dependence of the directing force on the electron transfer velocity, the absolute velocity constants can be analyzed in terms of the following equation: K = v exp [-ß (r-r0)] exp [ - (? G +?) 2/4? RT] where v is the velocity constant at the limit controlled by diffusion (1011 M- '-' '), r is the distance between the reactive and that produced in the activated complex, rn is the distance from the closest approximation of the reactive and the product, and ß describes the effect of the intermediate medium. The incorporation of the guanine donor inside the double helix imposes a finite distance through which the electron must make its way to the bound metal complex, that is, wtQ. However, if guanosine 5'-monophosphate (GMP) is used as an electron donor, direct collision of guanine with the metal complex (r = rn) is possible. For Fe (bpy) 33+ and GMP, the velocity constant measured by blocked flow is 2.6 x 103 M_1s-1. The known values of? for the related reactions they are on the scale of 1-1.5 eV, which gives a? G for et guanina par "*" 10 of 1.1 ± 0.1 V.

TABLE 1 Speed constants for the oxidation of guanine by Ru (bpy ^ in DNA oligomers fM-1 S'1) Oligomer sequence? rRUg (A) 1.2 x 10a (5'-AAATATAGTATAAAA) 1.7 (3 'TTTATATCATATTTT) GC pair 5.1 X 103 (5'-AAATATAgTATAAAA) GT no apar a2X -TTTATATIATATTTT) miéñtós 1.0 x 10 ° (5'-AAATATAGTATAAAA). GG does not mate- or X "(3'-TTTATATGATATTTT) 1.9 x 10" (5'-AAATATAGTATAAAA). GA does not mate- 0 A te'- TTTATAT TATTTT) while 1.8 x 10 * (5'-AAATATA (TATAAAA) chain 0 A individual 5.1 X 103 (5'-AAATATAGTATAAAA. 1.2 A. (3 * -TTTATATgtATTTT) a The DNA concentrations used to determine the rate constants were based on the moles of guanine nucleotides. bDistanda calculated opening of passage through the solvent. The calculated distances according to k / kss = exp [-ß? r], they were ß (H2?) = 3A-1 and kSs = 1-8 x 10"5M-1s-1.

Given that the speed constants are in relation to the guanine concentrates, the velocity observed for the non-pairing of GG has been normalized with respect to the other oligomers containing only one guanine. In Figure 2 are the cyclic voltammograms of Ru (bpy) 32+ in the presence of 5'-AAATATAGTATAAAA as an individual strand (C) and hybridized with its complementary strand (A). As in the case of GMP, r = rn for the individual chain, and the velocity constant of 1.8 x 10 $ "1 s-1 da? G (guanine + / n) = 1.1 V and? = 1.3 eV, which are According to the values of GMP oxidation, although there is a dramatic increase for the individual chain, only a slight increase is observed for the fully hybridized duplex at this scanning rate, resulting in a four-fold reduction in current after the Hybridization It is known that metal complexes such as Ru (bpy) 32 bind to the DNA in the minor groove, so that the velocity constant 150 times slower (1.2 x 103 M-1 s-1) for the oxidation of the Duplex should be the result of the distance between the guanine residue and the complex bound to the surface.When the metal complex is shortened in the minor groove, the guanine and the metal complex can not come into intimate contact, so the electron must make its way through the solvent that separates the resid uo de guanina and the metal complex. The displacement through the water is much less efficient than through non-polar means, and it is calculated that the value of ß for the water is approximately 3 A'1. The distance of the route can be calculated, therefore, according to : k / kss = exp (-ß? r) where? r is the change in distance in the duplex comparatively to the individual string. From this analysis,? R for the fully hybridized duplex is 1.7 A = Et large value of ß for the water suggests that important changes in the velocity constants of electron transfer will be effected by very small changes in the distance of travel, which in turn would reflect small alterations in the DNA structure. Also shown in Figure 2 is the voltamogram of Ru (bpy) 32 in the presence of the same duplex, where the base pair GC has been replaced by a non-pairing GA. The incorporation of the non-mating GA results in a two-fold increase in the general current compared to the true duplex, which results in a 16-fold change in the velocity constant (kQA = 1.9 x 104 M-1s-1). The velocity data for the individual chain, the fully hybridized duplex, and the three non-GX matings, are given in table 1. The calculated travel distances are also shown with respect to the individual chain. As expected, the guanine residue in the non-matings of G-purine is more accessible to the metal complex than in the non-pairing GT, where the two bases are still joined by two hydrogen bonds in an oscillating pair. However, the non-pairing GT still causes a change of 4 times the velocity constant, which is easily detectable. Therefore, the oxidon velocity constants follow the trend of G (single chain) > GA > GG > GT > GC. The ability to distinguish each of these non-pairings from each other provides the basis for the detection of hybridization sensitive to non-pairing that is sensitive even to non-pairings of individual base pairs in the pair of bases adjacent to the preselected base.

EXAMPLE 4 Modified bases to avoid oxidation in the probe chain: substitution of guanine for inosine Cyclic voltammograms were assembled using a working indium-tin oxide (ITO) electrode (area = 0.32 cm "), Pt wire counter electrode and an Ag / AgCI reference module. In Figure 5, a sample containing Ru (bpy) 32 at 25 μM and oligonucleotide at 75 μM dissolved in pH buffer of 50 mM Na phosphate (pH 7) with 0.70 M NaCl, was screened at 25 mV / s. In figure 6, a sample containing Ru (bpy) 32+ at 50 μM and 0.3 mM of 5'-GMP or 5'-IMP dissolved in aqueous solutons regulated in their pH and containing NaCl at 700 mM and pH regulator of Na phosphate at 50 mM (pH = 6.8, [Na +] = 780 mM) was screened at 2.5 mV / s from 0.0 V to 1.4 V. The scrutiny of the mononucleotides in the absence of Ru (bpy) 32 showed no appreciable oxidative current . A freshly cleaned ITO electrode was used for each experiment, and a bottom-up scrutiny of pH regulator was only subtracted from subsequent scrutiny. The second order velocity constants of the guanine oxidizer were determined by fitting the cyclic voltammetric data to a two-step mechanism using the DIGISIMMR software package. All parameters other than the oxidation rate were determined from voltammograms of the metal complex only in the same eroded. 5'-GMP was purchased from Sigma, and 5'IMP was purchased from U.S. Biochemical, and both were used without further purification. Oligonudeotides were prepared in the UNC Department of Pathology, and passed through a 3000 molecular weight cutoff filter to remove the mononucleotides. Purity was evaluated by reverse phase HPLC. The concentration was determined from the optical absorption at 260 nm, as described in Fasman, G.D. CRC Handbook of Biochemistry and Molecular Biology: CRC Press, Boca Raton, FL, 1975; Vol.1. The hybrid in Figure 5 was prepared by heating the complementary chains of 90 ° C for 5 minutes and cooling slowly to 25 ° C for 2 hours. These data indicate that guanine can be replaced by inosine in the probe chain to provide an inactive oxidation-reduction probe chain.

EXAMPLE 5 Passes modified to remove the eye on the chain in the probe 7- Deaza-Guanine This example is carried out essentially in the same manner as in Example 4 above, except that 7-deaza- is used. guanine as the modified base as an alternative for guanine to provide an inadivative oxidation-reduction probe chain. 7-deaza-guanine is oxidized at a rate of only 103 M_1 s-1, which is two orders of magnitude slower than guanine and is slow enough to provide an inactive oxidation-reduction probe chain.

EXAMPLE ß Detection using calf thymus DNA lyoado to the nylon membrane fiy to the ITO electrode The nylon film is cut into a ular shape, approximately 6 mm in diameter in order to fit in the electrochemical cell and cover the poron det ITO electrode exposed to the solution. For experiments in which only the cyclic voltammogram of the metal complex is obtained, the ITO reactor is first conditioned with a pH regulator. The nylon disk (without DNA) is then inserted into the electrochemical cell and 200 μL of a 200 μM metal complex solution is pipetted into the cell. For experiments with Os (bpy) 32+, a balance time of 6 minutes is used before the electrochemical analysis. For experiments with Ru (bpy) 32+, a balance time of 15 minutes is used before the chemical analysis. Cyclic voltammograms are checked using a PAR 273A potentiostat at a scanning speed of 25 mV / s. For DNA experiments, the nylon disk soaked with DNA is inserted into the electrochemical cell after conditioning the ITO eledrode in the proper pH regulator. 200 μL of a 200 μM metal complex solution at the appropriate pH regulator is pipetted into the cell, and a cyclic voltammogram is taken after the appropriate equilibrium time (6 minutes for Os (bpy) 32+ and 15 minutes for Ru (bpy) 32+ at a scanning speed of 25 mV / s The nylon disks are soaked for approximately 5 minutes in a 5.8 mM calf thymus DNA solution dissolved in water. from 5 minutes to 18 hours DNA is quickly associated (in a few minutes) with the nylon film, so that short immersion times are typically used.A low phosphate pH regulator is used under conditions of low salt content of Na at 50 mM (pH = 6.8, [Na +] = 80 mM.) Under conditions of high salt content, a pH regulator is used at 50 mM Na and 700 mM NaCl (pH = 6.8, [Na + ] = 780 mM.) The cyclic vottamogram of Ru (bpy) 32+ at the ITO-n lon electrode is shown in Figure 11. The dotted line shows the voltammogram when the nylon membrane is soaked in the calf thymus DNA before fixing it to the eledrode. There is a large catalytic current for the membrane marked with DNA that is equal to that observed in the soludon. The experiment shows that Ru (bpy) 32+ diffuses freely in the nylon film, and that DNA diffusion is not required to carry out the catalytic current. Figure 11 also shows that a higher catalytic current is observed at low salt concentrations, due to the increased interaction between the mediator and the immobilized DNA.

Figure 12A shows the same experiment using Os (bpy) 32+ as the mediator. The osmium complex does not oxidize guanine, so any current increase observed in the presence of guanine would have to arise due to the preconcentration of the mediator due to DNA binding. In fact, the current for Os (bpy) 32+ is lower in the presence of DNA in the nylon slide than in the absence of DNA. The experiment shows that the increased current for Ru (bpy) 32+ when the DNA is bound to the nylon core is due solely to the catalytic reaction and not to a trivial binding difference. The effect of the salt concentration is shown in Figure 12B, and it is observed that it is comparatively small with the great effect observed for the catalytic reaction. By attaching the DNA to the fixed nylon membrane to the ITO electrode, it has been shown that DNA can be detected even in the mode where the DNA is not spreading, but the mediator does. This finding allows the DNA to be detected where the immobilized probes are sufficiently close to the electrode, so that the hybrids labeled with a probe reside in the diffusion layer of the mediator.

EXAMPLE 7 Detection of RNA bound to the nylon membrane fiia to the ITO electrode The experiment is carried out as described in Example 6, except that Bakers yeast tRNA (acquired from Sigma) is used instead of DNA of calf thymus. A disk of nylon film was soaked in a tRNA solution as described in Example 6. The cyclic voltammetry in Ru (bpy) 32+ is shown in Figure 13. As in the case of DNA, current is observed catalytic for both pH regulators with more current at low salt content. The difference observed in the current between the high and low salt concentrations is not as dramatic as that observed with the DNA in Example 6, because the tRNA does not bind cations as well as DNA and, therefore, the effects of the salts are less dramatic. The results in Figure 13 demonstrate that RNA can be detected in a manner identical to that for DNA, which occurs because both RNA and DNA contain guanine. Therefore, the chemical composition of the sugar-phosphate base structure does not affect the catalytic current. Based on this observation, single and double stranded DNA and RNA, DNA-RNA hybrids, as well as individual or duplex strands containing other modified base structures such as PNA's, carbocycles, phosphorothioates, or other ribose ligands can be detected. replaced.

EXAMPLE 8 RNA Detection For the quantitative detection of RNA, a DNA probe (or RNA, PNA or other alternative base template) is immobilized on a solid support. The probe can be modified to be inert to oxidoon-reduced by substitution of the guanines by inosine or 7-deaza-guanine in the probe chain. The immobilized probe is then contacted with a target RNA terminus (e.g., HIV or hepatitis C virus). The solid surface contains then an immobilized probe inert to oxidation-reduction with an RNA chain. The solid surface is then contacted with a solution of Ru (bpy) 32+, and the cyclic voltammogram of the mediator is measured. The catalytic current signals the hybridization event, and the magnitude of the current is used to quantify the linked RNA chain based on the cone number of guanines in the chain. For the detection of RNA non-matings, a DNA probe (or RNA, PNA or other alternative) is immobilized to a solid surface. The preselected base in the probe chain is more easily oxidized than the other bases. The surface is placed in contact with a target RNA solution, and then put in contact with a solution of Ru (bpy) 32+ or another mediator. The degree of hybridization (perfect mating, no mating or no mating) is then determined at the preselected base in the same manner as for DNA.

EXAMPLE 9 Detection of a preselected base sequence The method was carried out as described in Example 3. The cyclic voltammograms shown in Figure 14 demonstrate that the current due to 5'-G is much smaller than that for 5'- GG which is much smaller than that for 5-GGG. This remarkable increase in current is observed for both individual and duplex chains containing GG and GGG sequences. The increase in current is not simply due to the increase in the number of G's since, as shown in figure 15, the increase in current due to the addition of G's to the same chain is much smaller than if the G's are intermingled. Since 5'-G of GGG is much easier to oxidize than a single G, it is possible to select a mediator (with a lower oxidation-reduction potential) that is capable of oxidizing to GGG but not to G. In Figure 16 The cyclic voltammogram of Ru (4,4'-dimethyl-bipyridine) 32+ is shown together with repeat scans in the presence of the individual G oligonucleotide and the GGG oligonudeotide. As shown, the catalytic current is observed only in the presence of the GGG oligonudeotide. This example shows the ability to fine-tune the potential of the mediator, so that a more easily oxidized sequence can be detected in the presence of the background guanine. The same strategy can be applied to detect an individual synthetic base that is derived to make it more easily oxidized than guanine. The experiment shows that it is possible to decrease the potential of the mediator and still distinguish a base or sequence of bases that are more easily oxidizable.

EXAMPLE 10 Detection of a preselected guanine derivative in the presence of native background guanine The disodium salt of 6-mercaptoguanosine 5'-monophosphate (6-S-GMP) (6-S-GMP) is prepared by commercially available phosphorylation of 6-mercaptoguanosine (from Sigma). The phosphoryladon is carried out using POCI3 according to the procedure of M. Yoshikawa et al., Bull. Chem. Soc. Jpn. 42: 3505 (1969). The disodium salt of 6-S-GMP is purified by HPLC prior to voltammetric analysis. Cyclic voltammograms are carried out at high ionic strength as in the example of inosine-5'-monophosphate. The work eledrodo is an ITO with a Hybond N + nylon membrane fixed to the surface to avoid the direct oxidation of the 6-S-GMP. The counter electrode is a Pt wire. The reference electrode is Ag / AgCI. The speed of scrutiny is 25 mV / s.

The results of the cyclic voltammogram are shown graphically in Figure 17, where curve A shows Ru (4,4'-Me2-bpy) 32+ only with (4,4'-Me2-bpy = 4,4'-dimethyl- 2-2'-bipyridine). After the addition of 5'-GMP, no increase in the Ru (Mß2bpy) 3 + wave is observed; however, the addition of 6-mercaptoguanosine 5'-monophosphate (6-S-GMP) leads to a dramatic current increase (curve B). The maximum current in the presence of 5'-GMP) is identical to that in curve A. The data show that it is possible to detect 6-mercaptoguanine bases in the presence of fundamental native guanine.

EJ? MPLQ H Detection of DNA hybridization with the preselected base on the target chain Nylon membranes (Hybond N +, Amersham, 480-600 μg / cm.sup.2) are cut into circular shapes, approximately 6 mm in diameter. The nylon discs are placed in a concentrated solution of polycytidyl acid (available from sigma) in water and allowed to soak for 1 hour. The discs are then removed from the polycytidyl acid solution (poly [C]) and placed on Parafilm and allowed to dry. As the discs are dried, 15 μL of additional poly [C] solution is added to the films in three aliquots of 5 μL. The discs are allowed to dry completely. The dried nylon discs are then washed with the low salt pH regulator (50 mM Na phosphate, pH = 6.8, [Na +] = 80 mM) to remove any poly [C] that is not tightly bound during the removal procedure. As a control experiment, a disc impregnated with poly [C] is placed through an imitation hybridization method in which it is not exposed to any adidonat nucleic acid, but is exposed to all other steps of hybridization. The disk is placed in 400 μL of milli-Q water, heated at 48 ° C for 1 hour and allowed to cool to room temperature. The disk is removed from the water and washed in a pH regulator with a low salt content before the electrochemical analysis. The discs prepared in this manner represent the background scrutinies (A) in Figures 18 and 19. A disk impregnated with poly [C] is placed in 400 μL of a polyguanilic acid (available from sigma) in water solution, heated at 48 ° C for 1 hour and allowed to cool to room temperature. The disk is then removed from the polyguanilic acid solution (poly [G]) and washed in a pH regulator with low salt content before the electrochemical analysis. The calf thymus DNA (available from sigma) in water is denatured (melted) by heating at 90 ° C for 10 minutes. A disc impregnated with poly [CJ] is placed in the denatured calf thymus DNA soluton, heated at 48 ° C for 1 hour and allowed to cool to room temperature. The disk is removed from the calf thymus DNA solution and washed in a low pH salt buffer prior to electrochemical analysis. As a control, a nylon disc that has not been impregnated with poly [C] is also subjected to the same procedure. The binding and DNA detection of calf thymus by absorption in the nylon film (not by hybridization) is observed in the control membrane. The nylon disc treated as described above, it is inserted in the electrochemical cell, after the conditioning of the ITO electrode with the low salt pH regulator. 200 μL of a Ru (bpy) 32+ solution at 200 μM are pipetted into the cell and a cyclic voltammogram is obtained after an equilibrium time of 15 minutes. The speed of scrutiny is 25 mV / sec. The cyclic voltammogram is reported in Figure 18. The poly [C] probe sequence is immobilized on a Hybond N + nylon membrane and the hybridization protocol is carried out on the pH regulator ("imitation hybridization"). The membrane is fixed to the ITO working electrode and (A) a cyclic voltammogram of Ru (bpy) 32+ is obtained. The membrane is immersed in a poly (G) solution and the hybridization is carried out according to the same protocol. The Glyclic voltammogram of Ru (bpy) 32+ is then measured (B), and a large current increase is obtained due to the catalytic oxidation of the hybridized poly [G] target. As shown in Figure 19, the test is specific to the appropriate sequence. Figure 19 compares the voltammetry for the poly [C] membrane where the hybridization process is carried out in a pH regulator (A) or in a single chain strand DNA solution (B). Figure 19 shows that if the target sequence is not present, no current increase is obtained.

EXAMPLE 12 Detection of DNA in nylon-modified vitreous carbon electrodes Figure 20 shows the cyclic voltammogram (or "CV") of a vitreous carbon electrode with a nylon film fixed before (A) and after (B) of the immobilization. of DNA on the nylon film. The nylon membrane (Zeta-Probe, Bio-Rad, 80-100 μg / cm2) was cut into circular shapes, approximately 5 mm in diameter. The nylon disk as configured covers the surface of the glass carbon electrode and is held in place by a plastic sleeve. For experiments in which only the cyclic voltammogram of the metal complex is obtained, the primary vitreous carbon electrode was conditioned with a pH buffer of 50 mM Na phosphate with low salt content (pH = 6.8, [Na +] = 80 mM). The nylon disk (without DNA) was then fixed to the eledrode and 400 μL of a Ru (bpy) 32+ solution at 200 μM was pipetted into the electrochemical cell. A balance time of 15 minutes was used before the chemical analysis. Cyclic voltammograms were obtained using a PAR 273A pentiostat using a scanning speed of 25 mV / s. In a typical DNA experiment, the vitreous carbon eledrode is first acondidone in the pH regulator of sodium phosphate with low salt content. A nylon disk was soaked for approximately 5 minutes in a 5.8 mM calf thymus DNA solution dissolved in water. The disk was then removed from the solution and placed on the glass carbon electrode using the sleeve to hold it in place. 400 μL of Ru (bpy) 32+ at 200 μM were pipetted into the electrochemical cell and after a 15 minute equilibrium a cyclic voltammogram was obtained using a scanning speed of 25 mV / s. The foregoing is illustrative of the present invention and should not be considered as limiting thereof. The invention is defined by the following claims with equivalents of the claims to be included therein.

Claims (10)

NOVELTY OF THE INVENTION R E I V I N D I C A C I O N S

1. - A method of DNA sequencing, characterized in that it comprises the steps of: a) counting a DNA sample with an oligonucleotide probe to form a hybridized DNA, said oligonucleotide probe including a preselected base having a unique oxidation rate; b) reacting said hybridized DNA with a transition metal complex capable of oxidizing the preselected base in said oligonudeotide probe in an oxidation-reduction reaction, said oligonucleotide probe having a predetermined number of preselected bases; c) detecting said oxidation-reduction reaction; d) measuring the reaction rate of said oxidation-reduction reaction detected; and e) identify the paired base with the preselected base.

2. The method according to claim 1, further characterized in that said identification step comprises: i) comparing the measured reaction rate with each of the four different known oxidation-reduction reaction rates of the transition metal complex with a DNA having adenine, cytosine, guanine or thymine linked to the preselected base; and ii) determining which of said known oxidation-reduction reaction velocities is essentially the same as the measured reaction velocity.

3. - The method according to claim 1, further characterized in that said oligonucleotide probe includes adidonately a preselected second base having a unique oxidation rate, wherein the oxidation rate of said preselected second base is different from the oxidation rate of the preselected base.

4. The method according to claim 3, further characterized in that said detection step further comprises detecting the oxidation-reduction reaction of the transition metal complex with said preselected second base; wherein the measurement step further comprises measuring the reaction rate of said detected oxidation-reduction reaction of the transition metal complex with the preselected second base; and wherein said identifying step further comprises identifying the paired base with the preselected second base.

5. The method according to claim 1, further characterized in that it further comprises repeating steps a) to e) with a sufficient number of oligonucleotide probes having said preselected base at different sites to identify each base in said DNA sample.

6. An RNA sequencing method, characterized in that it comprises the steps of: a) contacting an RNA sample with an oligonucleotide probe to form a hybridized RNA, said oligonucleotide probe including a preselected base having a speed of unique oxidation; b) reacting said hybridized RNA with a transidon metal complex capable of oxidizing the preselected base in said oligonucleotide probe in an oxidation reaction. reduction, said oligonucleotide probe having a predetermined number of preselected bases; c) detecting said oxidation-reduction reaction; d) measuring the reaction rate of said oxidation-reduction reaction detected, and e) identifying the paired base with the preselected base.

7. The method according to claim 6, further characterized in that said identification step comprises: i) comparing the measured reaction rate with each of the four different known oxidation-reduction reaction rates of the transition metal complex with an RNA having adenine, cytosine, guanine or uracil linked to the preselected base; and ii) determining which of said known oxidation-reduction reaction rates is essentially the same as the measured reaction rate.

8. The method according to claim 6, further characterized in that said oligonucleotide probe further includes a preselected second base having a unique oxidation rate, wherein the oxidation rate of said preselected second base is different from the rate of oxidation. oxidation of the preselected base.

9. - the method according to claim 8, further characterized in that said detection step comprises adidonally stopping the oxidation-reduction reaction of the transition metal complex with said preselected second base; wherein the step of measuring further comprises measuring the reaction rate of said oxidation reaction, detected reduction of the transition metal complex with the preselected second base; and wherein said identification step further comprises identifying the paired base with the preselected second base.

10. The method according to claim 6, further characterized in that it comprises adidonally repeating steps a) to e) with a sufficient number of oligonucleotide probes having said preselected base at different sites to identify each base in said RNA sample.