MXPA98000050A - Electrochemical detection of nucleic acid hybridization - Google Patents

Abstract

A method for detecting a nucleic acid (e.g., DNA, RNA) containing at least one preselected base (e.g., adenine, guanine, 6-mercaptaguanine, B-oxo-guanine and 8-oxo-adenine) comprises: reacting the nucleic acid with a trans-metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; detecting the oxidation-reduction reaction and determining the presence or absence of the nucleic acid from the oxidation-oxidation reaction; reduction detected at the preselected base, the method can be used in a variety of applications, including DNA sequencing, diagnostic tests and quantitative analysis

Description

ELECTROCHEMICAL DETECTION OF LINE HYBRIDIZATION OF NUCLEIC ACID CfltIPO DE Lfl INVENTION The present invention relates to the hybridization and sec-enclosure of nucleic acid, and particularly to methods for qualitatively and quantitatively deleting the hydration of nucleic acid and sequencing methods of nucleic acid.

BACKGROUND OF THE INVENTION The detection of id RDN 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 involves the use of surface-bound DNA sequences that can be tested using an analytical response that indicates the hybridization of the oligornero bound at the surface to a sequence in the heterogeneous sample. These analytical methods generally include laser-induced phosphorescence that originates from an e-tag fixed covalently on the target DNA chain, which is not sensitive to vanishing from a base in the duplex a do 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 (199 :)); IJairis, Ageu. Chern.107: 356 (1995) and Noble, Analitical Chernis + ry 67 (5) ¡201A (1995) propose surfaces or "plants" for this application. In an alternative method, proposed by Hall, et al., Biochem and Molec. 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 the mating of a base in the DNA rnues + ra. The techniques for detecting mismatches of a base include studies of the chemical or enzymatic cut such as those proposed in the US patent. No. 5,194,372 to Nagai et al. However, these techniques are disadvantageous since they require more time and separation technology. The pa.te of E.U.A. No. 5,312,527 to M H-elson et al., Discloses a selective vol-aunetric 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, there remains a need in the art for a method to detect DNA hybridization, which includes a method for detecting base desquamations, 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 that contains at least one preselected base (e.g., adenine, guanine, 6-mercaptog-anma, 8-oxo-guamna and 8-oxo-aden). river). 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 DNA that is hidded(b) reacting the hydrophobic DNA with a transition metal complex capable of oxidizing a preselected base in the oligonucleotide probe in an oxidation-reduction reaction, wherein the oligonucleotide probe has minus one of the preselected bases, (c) detecting the oxidation-reduction reaction and (d) determining the presence or absence of hydrophobed 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 flow of electrons 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) reacting the hybridized DNA with a transition metal complex capable of oxidizing a preselected base in the probe. oligonucleotide 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 reaction of oxidation-reduction detected, (e) compare the reaction rate measured with the oxidation-reduction reaction rate of the transition metal complex with a DNA chain and then (f) determine whether the measured reaction rate is essentially the same as the rate of oxidation reaction -reduction of the transition-metal complex with DNA from a chain. In 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 sample handling carrying the plurality of DNA sample containers, (c) an oligonucleotide probe delivery means for supplying the oligonucleotide probe or 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 for detecting an oxidation-reduction reaction. As a fourth aspect, the present invention provides a second apparatus for detecting the hybridization of the AUN. The apparatus includes (a) a DNA sample container, (b) an oligonucleotide probe delivery means for delivering a plurality of nucleotide oligonucleotide probes to the DNA sample container, (c) a release medium. of transition metal complex 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) making contact between a rnues * <; of DNA and an oligonucleotide probe to form a hybridized DNA, wherein the oligonucleotide probe includes a preselected synthetic base having an oxidation potential an LCO, (b) carrying the reacted DNA 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 reaction. reduction, (d) determining 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 detailed description or.

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 of Ru (bpy) 32+ at 25 rnV / s in 700 thousand NaCl / 50 rnM of sodium phosphate pH regulator. The dotted line represents the voltamogram of 50 μM Ru (bpy) 32+ and 3.0 rnri of calf thymus DNA (nucleotide).

Figure 2 shows the v lane cyclic branches of Ru (b? Y) 3-2 + in the presence of 5 '-AAATATAGTATAAAA as a single chain (C) and has complementary strands (A and B). The speed of scrutiny is 25 rnV / s. (A) represents 25 μM of Ru (bpy) 32+ + 100 μM of fully double-stranded bridged DNA (in guanine nucleotides) (5 '-AAATATAGTATAAAA) (3'-TTTATATCATATTTT). (B) represents Ru (b? Y) 32+ with a duplex containing a GA mismatch (5 '-AAATATAGTATAAAA) (3'-TTTATATAATATTT), and (C) represents Ru (hpy) 32+, a single string that contains a nucleus io of guanine (5 '~ AAATATAGTATAAAA). Figure 3 is a schematic illustration of an illustrative apparatus which is useful for carrying out the methods of the present invention. Figure 4 is a schematic illustration of a detection method particularly advantageous for quantitative detection of DNA, wherein the preselected base is located on the target nucleic acid. Figure 5 shows the cyclic voltarnogranules of Ru (bpy) 32+ (25 μM) at a scanning rate of 25 rnV / 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 i [5 '~ TTTTATACTATATTT]. (C) With 75 μM of the hybrid of the oligomer of B and d C5'-GGGAAATATAGTATAAAAGGG]. Working electrode: indium oxide contaminated with tin. Reference electrode: Ag / AgCl. Counter electrode: Pt wire. The secondary structure of the C hybrid is indicated in the figure. Figure 6 shows the cyclic volnograms of (A) R? (Bpy) 32+ (25 μM), (B) R? (B? Y) 3 + (25 μM) with '-ososmonophosphate (0.3 rnM), and (C) Ru (bpy) 3 + ( 25 μM) with g'anosine 5'-rnonophosphate. The structures of mosi a and guanina 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 useful microelectronic device 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 cyclic voltamnograms using nylon-modified ITO electrodes, Ru (bp) 3 + (200 μM) in nylon soaked with DNA in high salt pH regulator (700 rnM of added NaCl), and Ru (bpy) 32+ (200 μM) in nylon soaked with DNA in low salt pH regulator (ie, no NaCl was added). Figure 12 shows the cyclic voltarnograms of 0s (bpy) 3 + (200 μM) using nylon-modified ITO electrodes soaked with pH or DNA legulator. Figure L2A shows the cyclic voltarnogram with P00 rnM of NaCl added. Figure 12B shows the vol cyclic vol without MaOl added. Figure 13 shows the cyclic voltammograms in 1T0 electrodes modified with nylon showing cyclic volt mo-grams of Ru (b? Y) 32+ (200 μM) in nylon soaked with pH regulator, Ru (b? Y) 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 '-AAATATAG "TATAAAA where n = 1 (G), 2 (GG), or 3 (GGG). The speed of scrutiny is 25 rnV / s. Figure 15 shows the cyclic voltammogram of Ru (b? Y) 32+ (25 μM) alone and with (100 μM in chains) of 5'AAATAT (AGT) "ATAAAA where n = 1, 2, or 3. The speed of scrutiny is 25 rnV / s. Figure 16 shows the cyclic voltarnogram of 25 μM of 4,4'-d? Rnet ib? Pd? Na) 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 rnV / s. Figure 17 shows the cyclic voltammogram of 0.20 mM Ru (4,4'-e-2 ~ bpy) 3 + in 50 M sodium phosphate buffer (pH 7) with 0.7 M NaCl at a test rate of 25 mV / s. Curve (A) represents Ru (4,4'- e2-l? Y) 32+ alone. Curve (B) represents R? (4, '-Me? -bpy) 32 + in the presence of 5'-monophosphate of 6-rcanecaptoguans? Na of 0.70 rnM. Figure 18 shows 200 μM cyclic voltarnograms of Ru (bpy) 32+ in ITO working electi to which a Hybond N + nylon membrane is fixed. The membranes are impregnated with CC pol and subjected to the hybridization protocol in pH regulator (A) and a concentrated solution of polyCG] (B). Figure 19 shows 200 μM cyclic voltarnograms of Ru (bα) 32+ in ITO working electrodes to which a Hybond N ** - nylon membrane is fixed. The membranes are impregnated with polyCC] 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 voltarnograms (scanning speed = 25 mV / s) of 200 μM Ru (bμy) 32+ on a glassy carbon electrode modified with nylon (A) without DNA or (B) after the adsorption of DNA to the nylon film.

DETAILED DESCRIPTION OF THE INVENTION The term "nucleic acid" is used herein to refer to any nucleic acid, including both DNA and RNA. The nucleic acids of the present invention are typically polymorphic acids; that is, individual nucleotide polymers that are covalently linked by 3 ', 5' phosphodiester bonds. Tenni.no "complementary nucleic acid" is used herein to refer to any nucleic acid, including oligonucleotide probes that specifically bind to another nucleic acid to form a hLbriate nucleic 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, hybridization of the RNA, 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 should be understood that the methods and apparatus of the present invention can be applied to other nucleic acids such as RNA. fl. 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 certain applications to amplify the DNA prior to contacting the DNA. 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. K? Oh, Arn. Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplification techniques include, but are not limited to, the polymerase chain reaction (including, for the amplification of RNA, the reverse transcpptase polymerase chain reaction), the ligase chain reaction, amplification of the chain shift, amplification on the basis of transcription (see D. Kuoh et al., Proc. Nati. Acad ci.U.A. 86, 1173-1177 (1989)), self-sustained sequence replication (or "3SR") (see 3. G? atelli 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) ), amplification based on nucleic acid sequences (or "NASBA") (see R. Leis, Genetic Engmeepng News 12 (9), 1 (1992)), the repair chain reaction (or "RCR") ( see R. Lewis, supra), and boomerang DNA amplification (or "BDA") (see R. Le? is, s? pra). 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. The 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 which will be detected under Hybridization conditions for which an extension product of each primer is synthesized and is complementary to each nucleic acid chain, with the primers sufficiently complementary to each chain of the specific sequence to be given to the others so that the extension product synthesized from each initiator, when it is separated from s? complement, may serve as a pattern for the synthesis of the extension product of the other primer, and then treating the sample under denaturing conditions to separate the extension products from the primer from their standards if the sequence or sequences to be detected are present. These steps are repeated cyclically until the desired degree of amplification is obtained. The 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 known 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. Ualker et al., Proc. Nati Acad. Soi. E.U.A. 09, 392-396 (1992); G. lall-er et al., Nucleic Acids Res. 20, 1691-1696 (1992). For example, the SDA can be carried out with a single amplification primer or a pair of amplification primers, with exponential amplification being achieved 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 oligonucleotide probe of the present invention) that hybridizes to the target sequence that will be amplified and / or detected. The flanking sequence, which serves to facilitate ligation of the restriction enzyme to the recognition site and which provides a DNA polymerase initiation site after which the restriction site has been cut, is preferably about 15 μM. to 20 nucleotides in length; the restriction site is functional in the SDA reaction (ie, the phosphorothioate ligands incorporated in the initiator chain do not inhibit subsequent cleavage - a condition that can be satisfied 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 known techniques. See, e.g., R. Ueiss, Science 254, 1292 (1991). In general, the reaction is carried out with two pair of oligonucleotide probes: a pair is linked to a chain of the sequence that will be detected; the other pair is linked to the other string of the sequence that will be detected. Each pair together completely overlaps the chain to which it corresponds. The reaction is carried out, first, by denaturing (e.g., separating) the chains from the sequence to be detected, then reacting the chains with the two pairs of oligonucleotide probes in the presence of a stable ligase. heat 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. Oligonucleotide Probes As noted above, the methods of the present invention are useful for detecting DNA hybridization. The first step of the procedure includes doing with * t act between a DNA sample and an oligonucleotide probe to form a hybridized DNA. Oligonucleotide probes which 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, rn? And preferably between about 8 and about 15 base . Oligonucleotide probes can be prepared which have any of a wide variety of base sequences according to techniques that are well known in the art. Suitable bases for preparing the oligonucleotide probes can be selected from naturally occurring nucleotide bases such as arsenic, cough, guanine, uracil and tynin; and non-naturally occurring or "synthetic" nucleotide bases such as 8-oxo-guanine, 6-rnercaptoguanma, 4-acet? lc? t? dma, 5- (carbox? h? drox? et? l) u ? dma, 2'-0-met? lc? t? d? na, 5-carbox? met? lam? no-rnet? l-2-t? opd? na, 5-carboxy? namurnomomethyl-? ridma, dihydro? ridine , 2'-0-rnet? Lpseudour? D? Na, (3-D-galactosilqueosma, 2 '-0-rnet? Lguans? Na, inosine, N6-isopropellanyl adenosine, 1-rnet? Iodenos? Na, 1-met ? lpseudour? dma, 1-met? ig? anos? na, 1-met? lmos? na, 2,2-d? met? iguanos? na, 2-methyladenosine, 2-rnet? lguanosma, 3-met? lc ? t? dma, 5- me tl ci td na, N6-rnet? iadenos? a, 7-me lguanosma, 5-rneti larninornetiiupd na, 5-rnetox? arn? nornet? i ~ 2-t? our? d? na, ß-D-hands lkeosma, 5-methox? carbon? lrnet? l ur'dina, 5-rnetox? ur?? d? na, 2-rnet? lt? o-N6-? so? enten? ladenos? na, N- ((9-ß-D ~ pbofu anos? l-2-rnet? lt? op? pn-6-? l) carbamo? l) treomna, N- ((9-ß-Dp bof? ranosil -pur? n-6- l) N-rnet? l-carbarno? l) threonine, methyl ester of upd? n ~ 5-ox? acetic acid, updin-5-oxoacetic acid, wibutoxosma, pseudoup dina, queo sina, 2-t? oc? t? d? na, 5-met? l-2-to? pd? na, 2-t? o? r? dma, 2-t? oupd? na, 5-rnet? lurd? na, N ~ ((9-ß-Dr? bofurans? l? ur? n-6-? i) carba? no? l Jtreonina, 2 '-0-rnet? l-5-rnetil? ridine, 2'- 0-rnet? I? Rd? Na, wibutosma and 3- (3-am? No-3-carboxiprop Dupdi na.) Any base structure of oligonucleotide, including DNA, RNA (although RNA is preferred is preferred). DNA), modified sugars such as carbocycles and sugars containing 2 'substitutions such as fluoro and rnetox.The nucleotide oligos can be oligonucleotides in which at least one or all of the mucleucleotide bridging phosphate residues are modified phosphates, such as rnetii phosphonates, rnethyl phosphonothioates, phosphoror-morpholide, phosphoropiperazidates and phosphoramidates (for example, any of the other interstitial bridging phosphate residues can be modified as described.) The oligonucleotide can be a "peptide nucleic acid". "such as the one described in P. Nielsen et al., Science 254, 1497-1500 (1991) .The only requirement is that the The oligonucleotide must possess a sequence at least a portion of which is capable of bridging to a known portion of the sequence of the DNA sample. It may be desirable in some applications to make contact between the DNA sample and a number of oligonucleotide probes that have different base sequences (e.g., when there are two or more target nucleic acids in the sample, or when a simple target nucleic acid is hybridized to two or more probes in a "sandwich" test).

C. Hybridization methodology The DNA (or nucleic acid) sample can be contacted 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 contacted with the oligonucleotide probe by solubilizing the oligonucleotide probe in a solution with the DNA sample under conditions that allow hybridization. Suitable conditions are well known to those skilled in the art (see, e.g., patent of E.U.A. No. 4,358,535 to Fal ow et al., And other references to US patents. citing 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 contacted with the oligonucleotide probe by immersing the solid support having the probe. olagonucleotide is spread over it in the solution it contains with 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 reacted with a suitable oxidizing agent that is capable of oxidizing a base preselected in the oligoontrol 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 (v.gr-., The 5 '-deoxipbon? Cleotide or 5' -pbonucleotide) exhibit rate constants above l0 * -is_1 using the catalytic reaction. Examples of suitable preselected bases include but are not limited to guanine, adenine, 8-oxo-guanine, and 8-oxo-aderuna, 8-bromo-g? Amna, guanosma, xanthosine, wiosma, pseudo? Ridma, 6- mercaptog? an? na, 8-rnercaptoguanina, 2-t? oxant? na, 6-t? oxant? na, 6-r? er? a? ur? na, 2-am? no-6? carbox? met? l- rnercaptopur? na, 2 -rnerca? topup a, 6-methoxyp? pna, 2-acet? larn? no-6-h? drox? upna, 6-rnet? lt? o-2-hidroxip? rina, 2-d? rnet lam? no-6-h drox? pur? na, 2? h? drox? pupna, 2-arnmopur? na, 6-am? no-2 -d? rnet? lal? l-pur? na, 2-t? oaden? na, 8-hydroxyademine and 8-methox? aden? na. Typically, the preselected base is selected from the group consisting of guanine, adenine, 6-rnercaptoguan? Na, 8-oxo-guan? na and 8-oxo-aden na, with guanine being preferred as a preselected base occurring naturally and the 6-ier-toguanma as the currently preferred synthetic pre-selected base. The oxidizing agent can be any charged molecule such as a cationic, ammonic or zw + eponic molecule which is reactivated with the preselected baee in? N single oxidation potential. In this way, the selection of the oxidizing agent will depend on the preselected base chosen in particular, and will be easily determined by those skilled in the art. Particularly preferred oxidizing agents include the transition metal complexes which are capable of transferring electrons from metal DNA to the preselected base so that the reduced form of the metal complex is regenerated, completing a catalytic cycle. Examples of transition metal complexes suitable for use in the methods of the present invention include, for example, 2+ (2,2'-b? P? Pdina) 3 ruthenium ("Ru (bpy) 3 +") , 2+ (4,4'-d? Met? L-2,2'-b? P? R? Dma) 3 ruthenium ("R? (Me2-py) 32, 2+ (5,6-d) ? met? ll, 10-phenanthroline) 3 ruthenium ("Ru (e2-fen) 32 / 2+ (2,2 '~ b? p? r? d na) 3 iron (" Fe (bpy) 32+ "), 2+ (5-chlorophenan * t rolma) 3 (iron (" Fe (-Cl ~ phen) 32+ "), + (2,2'-b ??? r? D? Na) 3 of osmium ("0s (bpy) 32+") 2+ (5-chlorophenantrol-na) 3 of osmium ("0s (5-Cl-phen) 32+"), i + phosphine of dioxoremo and i + pyridine of dioxoremo ("Re? 2 (py U1 +) - Some ammonium complexes useful as oxidizing agents are: Ruíbpy) ((SO3) 2 -bpy) 22- and Ruíbpy) ((CO2) 2 -bpy) 22" and some complexes z? Useful materials as oxidizing agents are: Ru (bpy) 2 ((SO3) 2-b? Y) and Ruíbpy) 2 ((CO2) 2 -bpy, wherein (803) 2-bpy2- is 4,4'-d? Sulfonate-2, 2 '-bipipdin and (CO2) 2"-bpy2- is 4, '-d? Carbox? -2.2' -bipipdma. Suitable substituted derivatives of pipdma, bipyridine and phenanthroline and their groups can also be used in complexes with any of the foregoing metals. Suitable substituted derivatives include but are not limited to 4-ami nope idine, 4-d? Rnet? L? R? Dma, 4-acet? L? R? D? Na, 4-nt ropyridine, 4, 4 '-d? A? Nmo-2, 2' -bipir dma, 5,5 '-d? A? N? No-2, 2' -bi pin di na, 6,6 '-d? Am? No -2, 2 '-bipyridma, 4, 4 '-dietylenediamine-2,2' -bipipdin, 5, 5 '-d? Et? Lend? Arn? Na-2, 2' -bipipdine, 6,6'-d? Et? Lend? Am? na-2, '-bipipdin, 4,4'-d? h? drox? l-2,2' -bi pin di na, 5,5'-d? h? drox? l-2,2'-b ? p? pd? na, 6,6 '-dihydroxyl-2,2' -bipindin, 4,4 ', 4"-tpa? no-2, 2' 2" -terp? r? d? na, 4, 4 ', 4"-tr? Et? Lend? Arn? N-2,2', 2" -terp? Pd? Na, 4,4 ', 4"-trih? Drox? - 2,2', 2" -terp? r? d? na, 4,4 ', 4"-tr? mtro-2, 2', 2" -terp? r? d? na, 4,4 ', 4"-tr? feml-2 , 2 ', 2"-terp? R? D? Na, 4, 7-d? Am? No-l, 10-phenanthroline, 3, 8-d? Arnmo-l, 10-fenant rolina, 4.7- 1 d? et? iend am? na-1, 10-fenant roliría, 3, 8? d? et? lend? arnma-l, 10 - * (enantroli na, 4, 7-d? h? dro? ll, 10 -fenantrol, 3, 8-? h? drox? l-1, LO-feriant rol na,, P-dm tro-L, 10-phenanthroline, 1, 8-dm? tro-1, 10-fenantrol ? na, 4, 7-d? phen? li, 10-phenanthroline, 3, 8-d? em 1-1,10-phenanthroline, 4, 7-d? sper-amm-l, 10- fenantrolma, 3,8-d? sperarnm-1, 10-phenantrol ina and d? p? r? oC3,2-a: 2 ', 2' -cJIfenazma The oxidizing agent can be reacted with the hybrid DNA of 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 reacted with the hybridized DNA sample under conditions sufficient to effect the selective oxidation of the preselected base.For example, the transition metal can be reacted with hybridized DNA solubilized by soluting the oxidizing agent in the solution containing The hybridized DNA is 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 reacted with the hybridized DNA by immobilizing the oxidizing agent on the same solid support and immersing the solid support in a low solution. sufficient 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 solvent suitable for solubilizing 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 ligated in the minor groove of the DNA, and in this way an intimate contact is made between the preselected base and the oxidizing agent by the double-helix structure of the double helix. (or triple). This protection of the preselected base residue creates the need for an electron tunnel through the solvent, which decreases the speed of electron transfer. The solvent accessibility vain with the nature of the nucleotide base that is paired with the preselected base. The tunnel distance can be calculated according to the formula: l-Vkes = exp (-ß br) where r is the change in distance in the duplex compared to the single chain, and ss is the rate constant for oxidation 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 electron transfer is significantly less-than the reorganization energy (?), A plot of RT against the driving force, corrected for work terms associated with the reactance range, produces a line straight with a tilt of 1/2, according to Marcus's theory. Based on Marcus's theory then, the absolute velocity constants can be calculated using the following equation: 1- - v expC-ß (r-ro) lexpt- (, G +?) 2/4 XRT3 in which ? ev is the velocity constant at the controlled diffusion limit (lO ^ Mi si), r is the distance between the reactant and the product in the activated complex, ro is the distance from the nearest approach of the reactant and the product and ß it is the influence of the intervening medium. Because the preselected base, as previously noted, is incorporated into the hybridized DNA, this imposes a finite distance through which the electron must pass to the oxidizing agent. In this way, r is not equal to ro. ß for the water is approximately Sfi-1. This relatively large value for ß indicates that significant changes in the electron transfer rate 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 baee paired with the preselected base, the baee paired with the preselected base affects the distance of the tunnel through which the electron must pass between the preselected base and the 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 can be detected according to any suitable means known to the person skilled in the art. For example, the occurrence of the oxidation-reduction reaction can be detected using a detection electrode to observe a change in the electronic signal which is indicative of the occurrence of the oxidation-reduction reaction. Typically, a detection electrode, responsive to the transfer of electrons between the oxidizing agent and the hybrid DNA, is contacted with the solution containing the hybrid DNA that reacts and the oxidizing agent. In addition to the detection electrode, a reference electrode and an auxiliary electrode are generally brought into contact with the solution (most of the current passes through the auxiliary electrode). Suitable detection electrodes 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 chloride electrodes.

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 includes i) measuring the reaction rate of oxidation-eduction, 11) comparing the measured reaction rate with the oxidation-reduction reaction rate of the transition metal with a single-stranded DNA, and then m) determine whether the measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded DNA. The step of measuring the reaction rate can be carried out by any suitable means. For example, the relative reaction rate can be determined by comparing the current as a function of scanning rate, probe concentration, target concentration, mediator-, pH-regulator, temperature, and / or electrochemical method. The oxidation-reduction reaction rate can be measured according to the suitable means known to the person skilled in the art. Typically, the oxidation-reduction reaction rate is measured by determining the electronic 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 electrical signal that is generated, in order to provide a measurement of the oxidation-reduction reaction rate of the hybrid DNA reaction and the oxidizing agent. The electronic signal may be characteristic of any electrochemical method, including cyclic voltametry; Normal pulse, chronoamperornetria, and square wave voltammetry, with cyclical voltarnetry being the current pre-ida 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 bae in the DNA, either hybrid or single chain, affects the oxidation-reduction reaction rate between the oxidizing agent and the preselected base. . Therefore, hybrid DNA exhibits an oxidation-reduction reaction rate different from that of single-stranded DNA. The presence or absence of hybrid DNA in the preselected base can be determined by determining whether or not the measured oxidation-reduction reaction rate is the same as the oxidation-reductive 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 ligature 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 matches <; on the preselected base. For example, the oxidation-reduction reaction rate for the paired oxidation of guanine differs from the oxidation-r-eduction reaction rate for the oxidation of guanine paired with cytosine, which in turn is different from the velocity of oxidation-reduction reaction for the guanine-paired guanine oxidation, which is also different from the oxidation-reduction reaction rate for guanine oxidation paired with tyrosine. More specifically, the oxidation-reduction reaction rates for guanine oxidation follow the trend in which guanine from a single chain is greater than that from guanine paired with adam, which is greater than that from guanine paired with guanine, which is greater than that of guanine paired with timma, which is greater than that of guanine paired with cytokine. Therefore, the methods of the present invention are useful for detecting mismatches of a single base pair in the preselected base or in the base pair adjacent to the preselected base. Advantageously, the distinction between the rates of oxidation-reduction reaction of the oxidation of the preselected base when pairing with each of the different bases of natural occurrence, also allows the 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, 11) 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 also, cytosm, guanine, or thymine attached to the preselected base, and m) determining which of the known oxidation-reduction reaction rates is essentially the same as the measured reaction rate. The reaction rate 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 can be measured according to the same techniques with a DNA having adenine, cytosine, guanine or tintine attached to the preselected base, in such a way that these reaction rates are known. The measured reaction rate of the oxidation-reduction reaction of the oxidizing agent with the hybrid DNA can then be compared to the known oxidation-reduction reaction rates of the oxidizing agent with DNA having also, cytosine, guanine or tyramine attached to the preselected base. For example, the base paired with the preselected base is determined by determining the pairing of the known base that has the oxidation-reduction reaction rate essentially equal to the measured reaction-reduction reaction velocity.

F. Determination of the sequence of fl N The present invention also provides a method of determining the DNA sequence comprising a) contacting a DNA sample with an oligonucleotide probe to form a hybrid DNA; the oligonucleotide probe includes a preselected synthetic base having an oxidation potential umco; b) reacting the hybrid DNA with an oxidizing agent such as a transition metal complex, capable of oxidizing the preselected synthetic base in the nucleotide probe in an oxidation-reduction reaction, the oligonucleotide probe has a predetermined number of pre-selected synthetic bases; c) detecting 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 contact step 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 above in the preend and other synthetic bases known to the person skilled in the art. The only limitation is that the synthetic base must possess a unique oxidation potential compared to the oxidation potentials of the four bases that occur in nature, namely, adenine, cytosine, guanine and tine. 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 includes the steps of (i) comparing the reaction rate measured by each of the four different known oxidation-reduction reaction rates of the oxidizing agent with the DNA that has adenine, cytosine, guanine or tynine bound to the preselected synthetic base; (11) Determine which of the known oxidation-reduction reaction rates is essentially the same as the measured reaction rate. In another embodiment, the oligonucleotide probe also includes a preselected second synthetic base. The second preselected synthetic base has a unique oxidation potential that is different from the oxidation potential of the pre-selected synthetic base. In this embodiment, the step of detecting the oxidation-reduction reaction of the oxidizing agent with the preselected base also includes 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 rate also includes measuring the oxidation-oxidation reaction 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 synthetic base pf-β-sel also includes identifying the paired base also with the second preselected synthetic base. In accordance with this embodiment, the oxidation-reduction reactions of both preselected bases can be detected such 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, the DNA sequence can be determined by repeating the steps of the above method with a sufficient number of different probes of the oligonucleotide which has 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 includes at least one of the preselected synthetic bases, and the synthetic base is located in a different site and calculated along the sequence of the probe in each oligonucleotide probe. In this way, the repeated detection of the oxidation-reduction reaction of the hybrid DNA with an oxidizing agent, the measurement of the oxidation-reduction reaction rate, 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.

T. Apparatus The present invention also provides a useful apparatus for carrying out the methods of the present invention. One of these illustrative examples is shown schematically in Figure 3. In general, the apparatus comprises a plurality of containers 10 of m? Es * < DNA 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 delivery means for releasing the oligonucleotide probe in each of the DNA master containers, and a corresponding reservoir of liquid 15, Supply line 16 and valve 17 serve as an oxidizing agent release means to release the transition metal complex to each of the plurality of DNA sample containers. A probe assembly 20 including a lead 21 and a probe 22 serves as an oxidation-reduction reaction detecting means to detect 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 release means and the oxidizing agent release means for release the respective reagents in them. After the release of the reagent, 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 on the oxidation-reduction reaction. With the probe 22 additional electrodes are taken, necessary to carry out the cyclic voltammogram. 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 flasks, 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 the person skilled in the art, who 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 delivery means should allow sufficient contact between the DNA sample and the oligonucleotide probe ba or 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, in accordance with 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 detection electrodes and reference electrodes were described hereinabove with reference to the methods of the present invention. Preferably, the electrodes are in electronic communication with a means to measure the oxidation-reduction reaction rate. Suitable means for measuring the oxidation-reduction reaction rate 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 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); Bams, Agnew. Chem. 107: 356 (1995); and Noble, Analytical Chernistry 67 (5): 21 (1995), the descriptions of which are incorporated herein by reference in s? whole. As indicated above, the DNA sample container can be any suitable container known to the skilled person < hn Matter. The oligonucleotide probe release means is preferably a solid support having a plurality of oligonucleotide probes immobilized on the same, which is capable of releasing the probes to the i-NAR test container. For example, in accordance with one embodiment, the solid support which contains the plurality of oligonucleotide probes immobilized thereon is contacted with the DNA sample within the DNA sample container ba or conditions sufficient to allow the 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 above in the present. The preferred oxidizing agent releasing means comprises a solid support which has the oxidizing agent immobilized on the same. According to 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 performing 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 of a variety of DNAs, including pathogens, viruses, and the like. .

H. Detection of ñRN Hybridization, FlRN Sequencing, and Detection of FlRNA Disappearance. RNA hybridization detection methods, RNA sequencing methods, and RNA clearance detection methods are also described herein. Useful RNA for carrying out these methods includes, but is not limited to, ribosomal RNA, transfer RNA or genomic RNA (for example, 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) 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 oligonucleotide probe in an oxidation-reduction reaction, the oligonucleotide probe has 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 preselected 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 nucleotide probe in an oxidation-network reaction, the nucleotide wave probe has at least one of the preselected bases; (c) detect the oxidation-reduction reaction; (d) Edir- the reaction rate of the oxidation-reduction detected; (e) comparing the reaction rate measured with the rate of oxidation-reduction reaction of the transition metal complex to a single-stranded RNA; and then (f) determining whether the measured reaction rate is the same as the rate of oxidization-reduction reaction of the transition metal complex with single-stranded RNA. An RNA sequencing method comprises: (a) contacting an RNA sample with an oligonucleotide probe to form a hybrid RNA, the oligonucleotide probe includes a preselected base having a unique rate of oxidation; (b) reacting the hybrid RNA with a transition metal complex capable of oxidizing the preselected base in the oligonucleotide probe in an oxidation-reduction reaction, the oligonucleotide probe has a predetermined number of preselected bases; (c) detecting the oxidation-reduction reaction; (d) determining the reaction rate of the oxidation-reduction reaction detected; and (e) identify the base paired with the preselected base. The oligonucleotide probes, hybridization methodology, oxidation agents, detection of oxidation and reduction reactions, and apparatuses useful for carrying out these methods are essentially as indicated in the previous sections AH, adapted for use with RNA as the sample. of nucleic acid, in accordance with principles known to the person skilled in the art (for example, uracil replaces tynin as 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 electrochemical signal, and this signal is much weaker for double-stranded DNA. These methods advantageously exhibit a high structural sensitivity, and can resolve a single base mismatch. Therefore, these methods are particularly advantageous for DNA cleavage. However, two disadvantages of these methods are that: (a) there is a negative signal that goes from the probe chain to the hybrid, and (b) there is no signal amplification. The following techniques provide solutions to this problem. In addition, 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 the same, in which the target nucleic acid contains by itself, is also described herein. at least one preselected base. In contrast to the methods described above, in the present method the preselected base is located on the target nucleic acid, instead of on the oligonucleotide probe. The method can be carried out in a test sample containing the target nucleic 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, bacterial 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 eucanotic bacterial, protozoan, physical, protist, etc.), depending on the particular purpose of the test. The rnueetra can be treated or purified before carrying out the present method in accordance with techniques known or obvious 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) contacting the test sample with an oligonucleotide probe that specifically binds to the target nucleic acid to form a hybrid nucleic acid; (b) contacting the hybrid nucleic acid with a transition metal complex which oxidizes the preselected base in an oxidation-reduction reaction; (c) detecting the presence or absence of the oxidation-reduction reaction associated with the hybrid nucleic 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 hybrid nucleic acid test sample, 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 provided free in solution, and other means can be provided to separate the hybrid nucleic acid from the sample (for example by a nucleic acid buffer that binds to the oligonucleotide probe, or by an interaction of a biotin-avidin binding, in which the biotome is bound to the oligonucleotide probe and the 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 preferably at least 50 or 100 nm of the preselected base than the oligonucleotide probe. A greater current increase is advantageously obtained when the target nucleic acid contains more of the preselected base than the oligonucleotide probe. Optionally, but preferably, the oligonucleotide probe is free from the preselected base, or is at least essentially free from the preselected base (i.e., contains sufficiently less of the preselected base so that the probe signal does not interfere with it, m is mistakenly turned into a signal from the target nucleic acid). Where a sequence of naturally occurring bases which hybridizes conveniently with the target nucleic acid is not available, the strategy of employing alternative bases which are inactive 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 (for example, only A, T, and C) can be used. The cyclic voltarnogram of R? (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 overlapping paired base regions or in hanging regions, or both (as illustrated in Figure 4 by means of a "G" adjacent to the target nucleic acid chain), if the target nucleic 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 approximately provide 1,000-fold amplification per hybridization event. For example, in a preferred embodiment, the test for the preselected base on the target chain includes the immobilization of the probe chain (preferably inactive to the oxidation) on a solid surface oriented near the surface of the electrode, which provides a base signal ba a when scrutinized in the presence of the mediator. The solid surface then contacts a solution of the target chain, which contains the preselected base. If hybridization occurs, the target chain will now be close to the electrode, and an increase in current will be detected. 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., R? (Bpy) 33+), can be determined from the cyclic voltarnogran (or other electonic signal). through digital simulation. Under most conditions, this reaction follows the second-order kinetics, in which velocity = H_Rui bpy) 3 + HADN], where k is the velocity constant that is specific to the particular probe-target hybrid, CRu (b? y) 32 + 1 is the concentration of the oxidizing agent, and CADN] is the concentration of the hybrid (which could be a DNA-RNA hybrid). If they are known and CRu (b? Y) 32+], 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 solutions 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. Holodniy et al., 3. Virol. 69, 3510-3516 (1995); 3. MeLlors et al., Science 272, 116-1170 (1996). The oligonucleotide probes, the hybridization methodology, the oxidizing agents and the oxidation-reduction reaction methods, that of * < tion of oxidation-network reactions, and the apparatuses useful for carrying out these methods are as indicated in sections A-H above. 3. Alternative bases that are inactive for oxidation-reduction A disadvantage of the method described in section H above is that the oligonucleotide 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 substitute guanine (ie, a base that, like guanine, has a higher binding affinity for cytokine than the other bases in a double strand of nucleic acid) in the probe chain, but not oxidized by the oxidizing agent ba or the applicable reaction conditions. Examples of these alternative bases when guanine is the preselected base are inosine and 7-desazagamina. In this way, a method of detecting a target nucleic acid in which the nucleic acid contains at least one preselected base and the capture nucleic acid or probe contains inactive alternative bases to the oxidoreduction comprises: a) contacting the acid target nucleic acid with a complementary nucleic acid that specifically binds to the target nucleic acid to form a hybrid nucleic acid; b) reacting the hybrid nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation-eduction reaction; c) detecting the oxidation-reduction 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 cytokine (which ordinarily would bind with guanine in the complementary nucleic acid), then the complementary nucleic acid contains an alternative base that binds to the cytochrome in hybrid nucleic acid. The alternative base can be separated from the group consisting of inosine and 7-deazagiene ana. The reaction step comprises typical of the reaction of the transition metal complex with the nucleic acid or conditions sufficient to effect the selective oxidation 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 apparatus useful for carrying out these methods are as indicated in sections AI previous K. Polymerization of the preselected base with terminal transferase. An alternative embodiment of the method described in section H above includes elongating the target nucleic acid with terminal transferase to provide on it additional bases 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 nucleic acid, the oligonucleotide probe has terminal ends that stan 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 a prod? c * < or of extension of the target nucleic acid, the extension product comprises the preeleccalated base; c) contacting the oligonucleotide probe with a transition metal complex that oxidizes the preselected base in an oxidation-reduction 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) antenore.

The separation can be carried out by use of an immobilized probe, or the probe can be provided free in solution, as discussed in section H above. Oligonucleotide probes, hybridization method, oxidation-reduction reaction methods and oxidizing agents, detection of oxidization-eduction reactions and useful devices to carry out these methods, they are as indicated in sections A-1 above.

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 nucleic 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 binds specifically to the target nucleic acid; b) contacting the test sample with the capture probe to form a hybrid nucleic acid; c) contacting an oligonucleotide signal probe with the hybrid nucleic 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? n hybrid nucleic acid sandwich; d) contacting the hybrid nucleic acid sandwich with a transition metal complex that oxidizes the preselected base in an oxidation-eduction reaction; e) detecting the presence or absence of the oxidation-reduction reaction associated with the hybrid nucleic 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 polynephore bead, a plate or the inner surface of a well of rnicrotid plate). ? lo), 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 ar Ran i and others. Oligonucleotide probes may contain a polymer forming unit, as described in U.S. 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, Climcal Chern. 39, 725-726 (1993). A mediated polypeptide that binds to the oligonucleotide capture probe for immobilized polynucleotide, as described in US Pat. No. 4,751,177 for Stabinsky. The oligonucleotide probe may be attached to a member of a specific binding pair (eg, b otma), and the hybrid nucleic acid eigenvalue may be separated from the test sample by means of a second binding interaction with the another member of the binding pair, which is immobilized on a solid support (e.g., avidma), as described in R. Goodson, EPO Application 0 238 332: U. Harpson, EPO Application 0 139 489 , and N, Dattagupta, Application EPO 0 192 168. The oligonucleotide probes, the hybridization methodology, the oxidation agents and the oxidation-reduction reaction methods, the detection of oxidation-reduction reactions, and the apparatus that is used to carry out these methods, are given 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 detected even in the presence of a background signal produced from the oxidation of guanine. Because the detection of non-matings 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 faster than the other four bases. The present invention provides an oligonucleotide probe useful for the electrochemical 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 s? Bstituent of the 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 umon complement of the same. Specific examples of said oligonucleotide probes, and nucleotides useful for the preparation thereof, are the compounds of formula II: wherein: Ri is HO-P (O) (0H) -0-, a nucleotide, or an oligonucleotide; R2 is -H, a nucleotide or an oligonucleotide; R3 is -H, -0H, halogen (for example, fluorine, chlorine), alkoxy (for example, C1-C4 alkoxy, such as methoxy or ethoxy), arnino, or azido; and R * is -0- 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 the formula TI, Ri is HO-P (O) (OH) -O-. In another preferred embodiment of the compound of formula T, R is -H. When Ri is a nucleotide or an oligonucleotide gone, the phosphodiester binding is par-to the terminal 3 'end. When R2 is a nucleotide or oligonucleotide, the Ligature phosphodiester is for the 5 'terminal end. The compounds of 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 T 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-mercapto guanosine 5'-rnonophosphate (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 conductive substrate having an active surface formed on the ground; and (b) a polymer layer connected to the active 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 to the nucleic acid bound to the polymer). The conductive substrate may be a metallic substrate or a non-metallic substrate, including substrates which would be conductive (eg, gold, glassy carbon, tin oxide impuritated with indium, etc.). The conductive substrate may be any physical form, such as an elongated probe having an active surface formed on an extro or the same, 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 active surface, evaporating a solution of the polymer onto the electrode, or electropolishing. Exemplary polymers include, but are not limited to, nylon, t cellulose, polystyrene and poly (milp ridine). The thickness of the polymer layer is not critical, but it can be 100 Angstroms (fl) up to 1, 10 or even 100 microns. The electrode can be used in essentially all of the methods described in the above sections A-M. Thus, in general, the present invention provides a method for detecting a nucleic acid, said nucleic acid containing at least one preselected base, the method comprising: (a) contacting a sample containing said nucleic acid with an electrode, electrode comprising a conductive substrate having an active surface formed therefrom and a polymer layer as described above in relation to the active surface; (b) reacting the nucleic acid with a metal complex of oxidation capable of oxidizing the preselected base in an oxidation-eduction reaction; (c) detecting said oxidation-reduction reaction by measuring the current flow through said electrode; and (d) determining the presence or absence of the nucleic 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 microelectromechanical device for the electrochemical detection of a nucleic acid species in the methods described above, comprises a heterogeneous romeo substrate having first and second opposing surfaces; a conductive electrode - on the first surface; and an oligonucleotide capture probe immobilized on the first surface adjacent to the conductive electrode. The capture probe is separated sufficiently close to 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 hl on the adjacent electrode. In the preferred embodiment shown in FIGS. 9 and 10, a myeloelectric 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 separate electrodes. . By providing a plurality of separate loop probes, differing from each other, each with an associated electrode, a single compact device is provided which can detect several different hybridization events. Each electrode is electrically connected to an appropriate contact 23, so that the device can be connected or otherwise operatively associated with the necessary electronic equipment to carry out the steps of detection and determination of the methods described in the present invention. The nucleic acid can be immobilized selectively at the appropriate site on the microelectromatic substrate by known techniques. See, for example, the U.S. Patent. No. 5,405,783 by Pirrung and others. The microelect romeo substrate can be a semiconductor (for example, silicon), or non-semiconductor materials that can be processed using conventional microelectronic techniques (eg, glass). The electrode may be metallic or a non-metallic conductive material, such as polycrystalline silicon. The electrode can be formed using conventional microemotional processing techniques, such as etching with deposition acid. Several are romect inert structures and suitable manufacturing techniques are well known to the experts on the art. See, for example, S.M. Sze, VLSI Technology (1983); S.K. Ghandhi, VLSI Fabncation Principies (1983). The following examples are provided to describe the present invention, and should not be considered as limiting the same. In these examples, crn2 / s means square centimeters per second. M stands for molar concentration, -is-1 means per-second rnoles, eV stands for electrovolts, V stands for volts, nrn stands for nanornetros, GMP stands for guanosma 5'-monophosphate, and ITO stands for tin oxide impurified indium electrode. Cyclic volcanogranols were combined with a potentiostaf or galvanostat EG + G Model 273A from Ppnceton Applied Research, in accordance with known techniques. TTO working electrodes 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, St llwater, Minnesota 55082-1234 USA Nylon film is available as HYBOND-N + nylon membrane, catalog number RPN 1210B, from Arnersharn Corp, 2636 Clearbrook Drive, Arlington Heights, IL 60005 USA.

EXAMPLE 1 Measurement of the cyclic voltamogram of Ru (bpy) 32+ The cyclic voltamograms of Ru (b? Y) 3 +, 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 voltarnétpco scan. The voltarnetra of all DNA redox pair must be analyzed in terms of a quadratic scheme that relates the bound and unbound forms because the DNA diffusion coefficient is much smaller (ie, 2.0 x 10-7 crn2 / s) than that of the metal complex (8.0 x LO-6 cm2 / s). This phenomenon usually leads to run dramatically diminished entities for the linked; however, at high enough ionic strength [Na +] = 0.8 M), the binding of the rnetal 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.

R? (B? Y) 32+ »Ru (bpy) 33+ (E) Ru (bpy) 33+ + DNA» - R? (Bpy) 32+ + ADN0? (OR EXAMPLE 2 Analysis of cyclic voltammograms Cyclic voltarnograms were analyzed by adjusting the complete current potential curves, subtracting the background, using the data analysis package DTGISIMMR. The input parameters were E1 / 2 for the metal complex and the diffusion coefficients for the RNA complex and the DNA, all of which were determined by separate experiments. Therefore, the only parameter obtained from the adjustment was the second-order velocity constant for equation 2, k = 9.0 x lOSM-is "1. This same velocity constant was determined over a wide range of scrutiny velocities The rate constant for DNA oxidation by Ru (? Y) 33+ was confirmed in two separate experiments: First, square wave voltammograms were used to obtain a first order pseudo kobs for equation 2 by adjusting with the algorithm Je C00LMR The COOLMR algorithm uses an adjustment approach that is significantly different from DIGISIMMR, however, the ab * versus DNA plots were linear and gave a second order rate constant of k = 8.2 x 103 M- is-i, which matches the velocity constant obtained from the adjustment of cyclic voltammograms with DIGISirfiR.Second, authentic samples of bl were prepared and reacted Ru (bpy) 33+ with DNA directly in a blocked stream of rapid scrutiny. The global analysis of the dependent spectra for the time between 350 and 600 nrn showed that R? (B? Y) 33+ became cleanly Ru (bpy) 3 ~ 2 without any intermediate and a constant speed of 12 x 103 M-is-1 .. Thus, the rate constant for DNA oxidation by R? (Bpy) 33+ was established by means of two independent electrochemical measurements with dramatically different adjustment protocols and by means of a non-electrochemical flow technique. blocked with adjustment of the full visible spectra.

EXAMPLE 3 Analysis of cyclic voltammograms If the driving force for electron transference is significantly less than the reorganizational energy (X), a graph of the ln 1 of RT against the driving force (when correcting for useful terms associated with the approach of the reagents) should give a straight line with a slope of 1/2. The rate constants for DNA oxidation by metal (bpy) 33+ derivatives with different redox potentials are shown in Table 1 below. Since Marcus's theory describes the dependence of the driving force on the electron transfer velocity, the absolute velocity constants can be analyzed in terms of the equation sample: K = v expr-ß (r-ro)] exp [- (^ G + k) 2/4 > R? where v is the velocity constant at the boundary controlled by diffusion (1011 _is-i), r is the occurrence between the reactant and the product in the activated complex, ro is the distance from the closest approximation of the reactant 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 ligated metal complex, ie, r < »Ro. However, if guanosine 5'-monophosphate (GMP) is used as an electron donor, the collision of r-ecta from guanine with the metal complex (r = ro) is possible. Par-a Fe (bpy) 33+ GMP, the velocity constant measured by blocked flow is 2.6 x 103 -is-i. The known values of X for the related reactions are on the scale of 1-1.5 eV, which gives one for the guanine * 10 pair of 1.1 ± 0.1 V.

TABLE 1 Speed constants for oxidation of guanine by Rui bpy > 3 in RDN oligomers * The DNA concentrations used to determine the rate constants were based on the guanine nucleotide bases. b Calculated distance of opening of passage through the solvent. The distances calculated according to! - * / «,, -expU-ß £ > r3, were ß (H2?) = 3A ~? and ks «= 1.8 x J0-5M-iß- ?. Since the rate constants are in relation to guanine concentrations, the velocity observed for the non-pairing of GG has been normalized r-spec to the other oligomers containing only one guanine. In Figure 2 are the cyclic voltarnography of Ru (bpy) 32+ in the presence of 5 '-AAATATAGTATAAAA as an individual chain (C) and hybridized with its complementary strand (A).

As in the case of GMP, r * = ro for the individual string, and the velocity constant of 1.8 x 105 n-1, -1 da? .G (guamna + / o) = 1.1 V and? = 1.3 eV, which is in accordance with the GMP oxidation values. Even though there is a dramatic increase for the individual strand, only a slight increase is observed for the duplex fully hybridized at this scanning rate, resulting in a fourfold reduction in current after hybridization. It is known that metal complexes such as Ru (b? Y) 32 bind the DNA in the minor groove, so that the speed constant 150 times slower (1.2 x 103 for the oxidation of the duplex must 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 electronics must be opened It passes through the solvent that separates the guanine residue and the metal complex.The displacement through water is much less efficient than through non-polar media, and it is estimated that the value of ß for water is approximately 3. ß_1 The travel distance can be calculated, therefore, according to: where r is the change in distance in the duplex comparatively to the individual string. From this analysis, kr for the fully hybridized duplex is 1.7 fl. The large value of ß for water suggests that important changes in the rate constants of electron transfer will be effected by very small changes in the distance traveled, which in turn would reflect small alterations in the structure of the DNA . Also shown in Figure 2 is the voltarnogram of Ru (bpy) 32 in the presence of the same duplex, where the base pair GC has been replaced by? N non-pairing GA. The incorporation of the non-mating GA results in a two-fold increase in the general current compared to the authentic duplex, which results in a change of 16 times the speed constant (RGA - 1.9 x 10 M-iß_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 Lr with respect to the individual chain are also shown. As is to be expected, the guanine residue in the non-G-purine apparatuses is more accessible to the metal complex than in the non-mating GT, where the two bases are still joined by two hydrogen bonds in a couple-oscillating . However, the non-pairing GT still causes a change of 4 times the velocity constant, which is easily detectable. Therefore, the oxidation rate constants follow the trend of G (single chain) > GA > GG > GT > GC. The ability to distinguish each of these is not matched to each other, it provides the basis for the detection of non-pairing-sensitive hybridization that is sensitive even to non-pairing of pairs of individual baees in the base pair adjacent to the preselected base.

EXAMPLE 4 Modified bases to avoid oxidation in the probe chain: substitution of guanine by inosine Cyclic voltammograms were assembled using an indium-tin oxide (TTO) working electrode (area = 0.32 cm2), followed by a wire rod of Pt and an Ag / AgCl reference electrode. In Figure 5, a sample containing Ru (bpy) 32 at 25 μM and oligonucleotide at 75 μM dissolved in pH buffer of Na phosphate at 50 nm (pH 7) with 0.70 M NaCl, is screened at 25 rnV / s. In figure 6, a sample containing Ru (bpy) 3 + at 50 μM and 0.3 nm of 5'-GMP or 5'-IMP dissolved in aqueous solutions regulated in s? pH and containing NaCl at 700 rnM and phosphate pH regulator at 50 mM Na (pH = 6.8, CNa +] = 780 rnM) was screened at 2.5 mV / s from 0.0 V to 1.4 V. The scrutiny of the mononucleotides in the absence of R? (Bpy) 32 showed no appreciable oxidative current. A freshly cleaned ITO electrode was used for each experiment, and a background scrutiny of the pH regulator was only subtracted from subsequent scrutiny. The second-order speed constants of guanine oxidation were determined by fitting the cyclic voltammetric data to a two-step mechanism using the DIGISIMMR software package. All the parameters other than the oxidation rate were determined from voltanograms of the rnetal complex only at the electrode ism. The 5'-GMP was purchased from Sigma, and the 5'IMP was purchased from U.S. Biochernical, and both were used without further purification. Oligonucleotides were prepared in the UNC Department of Pathology, and passed through a molecular weight cut-3000 filter to remove the nucleotides. Purity was evaluated by reverse phase HPLC. The concentration was determined from the optical absorption at 260 nrn, as described in Fasman, G.D. CRC Handboo of Biochemistry and Molecular Biology; CRC Press, Boca Raton, FL, 1975; Vol.l. 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 mosine in the probe chain to provide an inactive oxidation-reduction probe chain.

EXAMPLE 5 Modified Bases to Prevent Oxidation in the Probe Chain: 7-Deaza-Guanine This example is carried out in essentially the same manner as Example 4 above, except that 7-deaza-guamna is used as the modified base as an alternative for guanine to provide a chain of inactive oduction-eduction probe. 7-deaza-uanine is oxidized at a rate of only 103 rl-ig-i, which is two orders of magnitude slower than guanine and is slow enough to provide an inactive oxidation probe chain-- reduction EXAMPLE 6 Detection using calf thymus DNA bound to the nylon membrane fixed to the ITO electrode The nylon film is cut into a circular shape, approximately 6 nm in diameter in order to fit in the electrochemical cell and cover the portion of the ITO electrode exposed to the solution. For experiments in which only the cyclic volume of the metal complex is obtained, the ITO electrode is first conditioned with a pH regulator. The nylon disk (s n DNA) is then inserted into the electrochemical cell and 200 μL of a solution of the metal complex at 200 μM is pipetted into the cell. For experiments with 0s (bpy) 32+ a 6 minute equilibrium time is used before the electrochemical analysis. For experiments with Ru (bpy) 3+, a balance time of 15 minutes is used before the electrochemical analysis. Voltamog cyclic branches are collected using a PAR 273A potentiostat at a scanning speed of 25 rnV / s.

For DNA experiments, the nylon disk soaked with DNA is inserted into the electrochemical cell after conditioning the ITO electrode at the proper pH regulator. 200 μL of a 200 μM metal complex solution in the appropriate pH regulator is pipetted into the cell, and a cyclic vortex becomes after the appropriate equilibrium time (6 minutes for 0s (b? Y) 3 + and 15 minutes for Ru (bpy) 32+ at a scanning speed of 25 mV / s.The nylon disks are soaked for approximately 30 minutes in a solution of calf thymus DNA at 5.8 mM in water. They investigated vain immersion times ranging from 5 minutes to 18 hours.The DNA is quickly associated (in a few minutes) with the nylon film, so that brief immersion times are typically used.With conditions of low salt content, uses a phosphate pH regulator of Na at 50 rnM (pH = 6.8, CNa +) = 80 rnM Under conditions of high salt content, a pH regulator solution of Na at 50 rnM and NaCl at 700 rnM is used (H = 6.8, CNa +] = 700 inM) The cyclic voltamogram of Ru (bpy) 32+ at the ITO-nylon electrode is shown in figure 11. The dotted line shows the voltammogram when the nylon membrane is soaked in the calf thymus DNA before attachment to the electrode. There is a large catalytic current for the membrane marked with DNA that is equal to that observed in the solution. The experiment shows that Ru (bpy) 3 + diffuses freely in the nylon film, and that the diffusion of the DNA is not required to carry out the catalytic current. Figure 11 also shows that a greater catalytic current is observed at low concentrations of salts, due to the increased interaction between the mediator and the immobilized DNA. Figure 12A shows the same experiment using 0s (bpy) 32+ co or the mediator. The osmium complex does not oxidize guanine, so that any increase in current in the presence of guanine would have to arise due to the preconcent of the mediator due to DNA binding. In fact, the current for 0s (b? Y) 32+ eß less in the presence of DNA in the nylon electrode than in the absence of DNA. The experiment shows that the increased current for Ru (b? Y) 32+ when the DNA is bound to the nylon electrode is due solely to the catalytic reaction and not to a trivial binding difference. The effect of the concentration of salts is shown in Figure 12B, and it is observed that it is comparatively small with the great effect that is observed for the catalytic reaction. By attaching the DNA to the fixed nylon membrane to the TTO 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 makes it possible to detect the DNA where the immobilized probes are close enough to the electrode, so that the hybrids marked with a probe reside in the diffusion layer of the mediator.

EXAMPLE 7 Detection of RNA bound to the nylon membrane fixed to the ITO electrode The experiment is carried out as described in Example 0, except that Bakers' yeast tRNA (acquired from Sigrna) is used instead of calf thymus DNA. A disk of nylon film was soaked in a tRNA solution as described in Example 6. Cyclic voltarnetry in the presence of Ru (bpy) 3 + is shown in Figure L3. As in the case of DNA, catalytic current is observed 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 Fjernplo G, because the tRNA does not bind cations as well as DNA and, therefore, the Effects of salts are less dramatic. The results in Figure 13 show that RNA can be detected in a manner identical to that for DNA, which happens 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 strand DNA and RNA, DNA-RNA hybrids, as well as individual or duplex strands containing other modified base structures such as PNA's, carbocycles, phosphorus loathates, or other 1? gadgets of ribose subsumed.

EXAMPLE 8 Detection of flRN For the quantitative detection of RNA, a DNA probe (or RNA, PNA or other alternative base structure) is immobilized on a solid support. The probe can be modified to be inert to oxidation-reduction by substitution of guanines by inosine or 7-deaza-guamna in the probe chain. The immobilized probe is then contacted with a target RNA solution (for example, HIV or hepatitis C virus). The solid surface then contains an immobilized probe inert to oxidation-reduction with an RNA chain. The solid surface is then contacted with a solution of Ru (bpy) 3+, and ina of the cyclic voltarnograin of the mediator. The catalytic current signals the hibr-ation event, and the magnitude of the current is used to quantify the linked RNA chain based on the known 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 oxidized more easily than the other bases. The surface is placed in contact with an RNA solution 71 target, and then put in contact with a solution of Ru (bpy) 32 + or another measurer. The degree of hybridization (perfect pairing, without pairing or not ap- praising) is then determined at the base p'-esel, as is done in the same way 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 less than that for 5' -GGG. This remarkable increase in current is observed for both single 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 less 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 voltarnogram of Ru (4,4 '~ d? rnet? lb? p? r? d? na) 3 + is shown along with repetitive scrutinizations in the presence of the individual G oligonucleotide and r * the oligoinucleotide of GGG. As shown, the catalytic current is observed only in the presence of the GGG oligonucleotide. This example shows the ability to fine-tune the mediator's potential, so that a more easily oxidized sequence can be detected in the presence of background guanine. The same strategy can be applied to detect- an individual synthetic base that is derived to make the rnas easily oxidized than guanine. The experiment demonstrates that it is possible to decrease the mediating potential and to distinguish even a base or sequence, ie bases that are more easily oxidizable.

EXAMPLE 10 Detection of a preselected guanine derivative in the presence of the native guanine in the background The disodium salt of 6-mercaptoguans? Na 5'- nonofosfato (6-S-GMP) (6-S-GMP) it is prepared by commercially available 6-rnercaptoguanosine phosphorylation (from Sigrna). Phosphorylation is carried out using POCI3 according to the procedure of M. Yoshikawa and otos, Bull. Chern. Soc. Jpn. 42: 3505 (1 69). The disodium salt of 6-S-GMP is purified by means of HPLC prior to an ionis voltametco. Lieos voltarnograms are carried out at high ionic strength as in the example of the? Na? Na-5 '-monophosphate. The working electrode is an ITO with a Hybond N + nylon membrane fixed to the surface to prevent direct oxidation of 6-S-GMP. The counter-electrode is a Pt wire. The reference electrode eß of Ag / AgCl. The scrubbing speed is 25 rnV / s. The results of the cyclic voltarnogram are shown graphically in Figure 17, where curve A shows Ru (4,4'- e2- p and) 32+ only with (4, 4 '- ß2-bpy = 4, 4' -d? rnet? l-2-2 '-bipindin). After the addition of 5'-GMP, no increase in the Ru (Me2 bpy) 32+ wave is observed; However, the addition of 6-? nerca? tog? anosine 5 '-mono phosphate (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 cur-va A. The data show that it is possible to detect 6-rnercaptoguanin baees in the presence of fundamental native guanine.

EXAMPLE 11 Detection of DNA hybridization with the preselected base on the target chain Nylon membranes (Hybond N +, Arnersham, 480-600 μg / cm) are cut into circular shapes, approximately 6 mrn < the diameter. The nylon discs are placed in a concentrated solution of citric acid 1 to co (available from sigma) in water and < -e let soak- for 1 hour. The discs are then removed from the polycytidyl acid (polyTC] solution and placed on Parafilm and allowed to dry. As the discs dry, 15 μL of additional poly [CU] 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 (Na phosphate at 50 rnM, pH = 6.8, CNa + 1 = 80 rnM) to remove the polyclor that is not tightly bound during the procedure. of removal. As a control experiment, a disc impregnated with polyCC] is placed through an imitation hybridization procedure in which it is not exposed to any additional nucleic acid, but is exposed to all subsequent hybridization steps. The disc is placed in 400 μL of rnili-Q water, heated at 48 ° C for 1 hour and allowed to cool to room temperature. The disc 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 polyCC] is placed in 400 μL of polyglycolic acid (available from sigma) in water solution, heat at 48 ° C for 1 hour and allow to cool to room temperature. The disk is then removed from the poLi guaní 1 a co (polifGJ) acid solution and washed in a pH regulator with low salt content before the olecular analysis. The calf thymus DNA (available by itself) in water is denatured (molten) by heating at 90 ° C for 10 minutes. A disc impregnated with polyCC] is placed in denatured veal or ti DNA solution, heat at 48 ° C for 1 hour and allow to cool to room temperature. The disk is removed from the calf thymus DNA solution and washed in buffer solution of ba or salt content before the electrochemical analysis. As a control, a nylon disc that has not been impregnated with polyc is also subjected to the same or procedure. The binding and detection of DNA from terner-a by absorption in the nylon film (not by hybridization) is observed in the control membrane. The tr-attached nylon disk, as described above, is inserted into the electrochemical cell, after the conditioning of the ITO electrode with the low salt pH regulator. 200 μL of a solution of Ru (bpy I3 + at 200 μM are pipetted into the cell and a cyclic vol arnograine is obtained after an equilibrium time of 15 minutes, the scanning rate is 25 inV / sec. Cyclic voltammogram is reported in Figure 18. PolitC] <The probe sequence is immobilized on a Hybond N + nylon membrane and the protocol for hybridization is carried out on the pH regulator ("imit cy hybridization"). The membrane is fixed to the ITO working electrode and a (R) cyclic volume of R (bpy) 32+ is obtained. The membrane is immersed in a polyTG solution and the hybridization is carried out according to the same protocol. The cyclic voltarnogram of Ru (b? Y) 32+ ee m of "Jespues (B), and a large current increase is obtained due to the catalytic oxidation of the given polyCG target]. As shown in Figure 19, the test is specific to the appropriate sequence. Figure 19 compares the voltarnetry for the poly-membrane] where the hybridization procedure is carried out in a pH regulator (A) or in a solution "ie single-strand calf thymus DNA (B). Figure 19 shows that if the target sequence is not present, no current increase is obtained.

EXAMPLE 12 Detection of flDN on vitreous carbon electrodes modified with nylon Figure 20 shows the cyclic voltammogram (or "CV") of a vitreous carbon electrode with a nylon film "Ja before (A) and after (B)" He immobilized DNA on 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 disc as it is 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 phosphate pH regulator at 50 nm with low salt content (? H = 6.8, CNa +3 = 80 rnM). The nylon disk (without DNA) was then fixed to the electrode and 400 μL and a solution of Ru (bμy) 32+ at 200 μM were pipetted to the elektrochemical cell. A 15-minute elapse time was used before the electro-analysis} u m? < "o Cyclic voltammograms were obtained using a pentios or PAR 273A using a scanning speed of 25 rnV / s In a typical DNA experiment, the vitreous carbon electrode is first conditioned in the pH regulator of sodium phosphate with low salt content.A nylon disc was soaked for about 5 minutes in a 5.8 inM calf thymus DNA solution dissolved in water.The disk was then removed from the solution and placed on the glassy carbon electrode using the cuff to hold it in place - 400 μL of Ru (bpy) 3 + at 200 μM were pipetted into the electrochemical cell and after a 15 minute equilibrium a cyclic voltarnogram was obtained using a 25 inV pacing rate. 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 00 equi alentes of the claims that are to be included in it.

Claims (5)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A method for detecting DNA hybridization which will: a) contact a DNA sample with a pair of oligonucleotide pairs to form a hybridized DNA; b) causing said hybrid DNA to be rotated with a transition metal complex capable of oxidizing a preselected DNA in said oligonucleotide probe in an oxidation-reduction reaction, giving the oligonucleotide probe having at least one such base preselected; e) detecting said oxidation-reduction reaction; d) determining the presence or absence of DNA hybridized to par-t r of said oxidation-induced reaction in the preselected base; and e) identifying the varied base with said preselected base or the paired base with the base adjacent to the Leccaonada prese base.
2. 2. The method according to claim 1, further characterized in that the step of determining further comprises the steps of: i) measuring the reaction velocity of said oxidation-reduction reaction, n) comparing said measured reaction rate with the oxidation-reduction reaction rate of the metal complex "transition with a single-stranded DNA; and «Jespues m) determine whether the measured reaction rate is essentially the same as the reaction rate of the oxidation-reduction of the transMion metal complex with single-stranded DNA.
3. 3. The method according to claim 1, further characterized in that said DNA sample is a sample of single-stranded DNA, said hybridized DNA is a duplex.
4. 4. The method according to claim 1, further characterized in that said oligonucleotide probe includes from about 4 to about 100 bases. 5. The method according to claim 1, further characterized in that said base proselecci nothing is guanine. 6. - The method according to claim 1, further characterized in that said pre-selected base is aderuna. 7. The method according to claim 1, further characterized in that said transition metal complex is selected from the group consisting of Ru (bpy) 32 + / Ru (Me2 ~ t) py) 32+, Ruí e? -phen) 32+, Fe (b? y) 32+, Feí 5-Cl-? hen) 32+, 0s (5-Cl-phen) 32+ and Re? 2 (? YU1 + 8.- The method according to claim 1, further characterized in that said reaction step comprises reacting transition metal complex with the sample of DNA hybridized low with "enough signals to effect the selective oxidation" of the preselected base 9. The method according to claim 1, further characterized in that it comprises the step of amplifying and hybridizing DNA before the step contact. S33 The method in accordance with the indication indication 9, in addition to this step of amplifying the DNA sample is carried out by chain reaction of polynuclease, amplification of chain displacement, reaction of ligase chain or amplification based on nucleic acid sequence. 11. The method of confidence with claim 2, further characterized by the fact that said step of editing the reaction rate of the oxidation-reduction reaction depends on the cyclic voltanogram of the leaction. 12. The method according to claim 2, further characterized in that said comparison step comprises comparing the cyclic voltammogram of the reaction "Jel transition metal complex with the hybridized DNA sample against the known cyclic voltammogram of the reaction of the transitional rnetal complex with single-stranded DNA. 13. The method according to claim 1, further characterized in that said oligonucleotide probe is immobilized on a solid surface. 14.- The "Jo" in accordance with the claim 13, further characterized in that said transition metal complex is immobilized on the solid surface. 15. The method in accordance with the claim 1, further characterized in that said identification step further comprises the steps of: i) measuring the reaction rate of said oxidation-reduction reaction detected, U 11) compare the reaction rate rne «ia < 1a of each of the four different known oxidation-reduction reaction rates of the transitional complex with DNA "tonataone, eitosis, gunin, or tint linked to 1 A preselected base; lai) determining which of said known oxidation-reaction reaction velocities is essentially the same as the measured reaction rate. 16. A method for detecting DNA hybridization comprising: a) contacting a sample of DNA with an oligonucleotide probe to form a hybridized DNA; b) reacting said flanged DNA with a transition metal complex capable of oxidizing a preselected base in said oligonucleotide probe in an oxidation-reduction reaction, said oligonucleotide probe having at least one of said pre-selected bases; c) detecting said oxidation-reduction reaction; d) measuring the reaction rate of said oxidation-reduction reaction; e) comparing the reaction rate measured with the rate of oxidation-reduction reaction of the transition metal complex with a single-stranded DNA; f) determining whether the measured reaction rate is the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded DNA; and g) identifying the varied base with said preselected base or the paired base with the base adjacent to the preselected base. 17. The method according to claim B5 16, also characterized popjue said preselected base is Guaní na. 10. The method according to claim 16, further characterized by "said base prese! eccionada is ado na. 19. The method of compliance with the claim 16, I also acter-Lied because that transitional metal complex is selected from the group consisting of Ru (bpy) 32+, Ru (Me2-t * py) 32+, RulMe2 -? Hen) 32+, Fe (b? Y) 32+, Fe (5-Cl ~ phen) 32+, Os (5-Cl-? Hen) 32+ and Re? 2 (? yU1 + 20.- The method according to claim 16, further characterized in that said reaction step comprises contacting said transition metal complex with the DNA sample under sufficient conditions? to carry out the selective oxidation of said preselected base. The method according to claim 16, further characterized because it comprises the step of amplifying said DNA before the reaction step. with the claim 21, further characterized in that said amplification step of the hybridized DNA is carried out by reaction of pol erase, amplification of chain shift, ligase chain reaction or amplification of nucleic acid base sequences. . 23. The method according to claim L6, further characterized in that said measurement step comprises measuring the cyclic voltammogram of said reaction. 24. The method of conformity in claim 16, further characterized in that said step of comparing comprises comparing the cyclic vol to the reaction of the transition metal complex with the DNA sample hybridized against the known cyclic voltammogram. The reaction of the transition metal complex with single-stranded DNA. 25. The method according to claim 16, further characterized in that said oligonucleotide probe is immobilized on a solid surface. 26. The method according to claim 25, further characterized in that said transition metal complex is immobilized on the solid surface. 27. The method of compliance with the claim 16, further characterized in that said base identification step (g) comprises i) comparing the measured reaction rate of each of the four different known oxidization-reduction reaction rates of the transition metal complex with a DNA that has adenine, cytosine, guanine or thymine linked to the preselected base; li) determining which of said known oxidation-reduction reaction rates is essentially the same as the measured reaction rate. 28. An apparatus for detecting DNA hybridization comprising: a) a plurality of DNA sample containers; b) sample handling means for carrying said plurality of DNA sample content; c) oligonucleotide probe assortment means to deliver said oligonucleotide probe to each of the DNA sample containers; d) assortment means < The transition metal complex is to supply said transition metal complex to each of a plurality of DNA sample containers; e) a detector for the oxidation-reduction reaction to detect an oxidation-reduction reaction; and f) means for measuring the oxidation-reduction reaction rate of said oxidation-reduction reaction detected. 29. The apparatus according to claim 28, further characterized in that said oxidation-reduction reaction detector comprises an electrode. 30.- The apparition in accordance with the claim 28, character- ized further because said means of assortments of the nucleotide probe comprises a solid surface having an oligonucleotide probe and immobilized on the same. 31. An apparatus for detecting DNA hybridization comprising: a) a DNA sample container; b) Assortment means "Je probe of ol? gon? cleot" Jo to deliver a plurality of oligonucleotide probe to said DNA sample container; c) Means "The complex of metal complex" The transition to supply said metal complex "Transition to the DNA sample container; and d) an oxidation reaction detector- 00 reduction to detect an oxidation-reduction reaction; and o) means --- to measure the reaction rate of oxidization-reduction of said oxy-on-reduetion reaction detected. 32. The apparatus according to claim 31, further characterized in that said reaction detector "reduction reduction" comprises an electrode. 33. The apparatus according to claim 31, further characterized in that said oligonucleotide probe assortment means comprise a solid surface having a plurality of oligonucleotide probes immobilized thereon, wherein each of the oligonucleotide probes is different from another. 34. The apparatus according to claim 33, further characterized in that said transition metal complex assortment means comprises a solid surface having a plurality of oligonucleotide probes and said transition metal complex immobilized on the bases. . 35.- A DNA sequencing method comprising: a) contacting 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 of said oligonucleotide probe in an oxidation-reduction reaction, said oligonucleotide probe having a predetermined number of bases. μselected; c) detecting said oxidation-reduction reaction J ?; d) measure the reaction rate of said reaction "oxidation-reduction detected; and e) identify- the paired base with the selected base. 36.- Flneth in accordance with the claim 35, further characterized in that said identification step comprises: i) comparing the measured reaction rate of each of the four different known oxidation-reduction reaction rates of the transition metal complex with a DNA having adenylate, cytosine na, guanine or ti mine linked to the preselected base; n) determining which of said known oxidation-reduction reaction rates is essentially the same as the measured reaction rate. 37.- The method according to the claim 35, further characterized in that said cleotide oligon 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 oxidation rate of the preselected base. 38.- The method according to claim 37, further characterized in that said detection step further comprises detecting the oxidation-reduction reaction of the transition metal complex with said pre-selected second base.; In addition, the measurement step further comprises measuring the reaction rate of said oxidation-reduction reaction "jected from the transition metal complex with the second preselected base, and wherein said identification step further comprises identifying base paired with second base prese! eccionada 39.- The conformity rnetoiJon the claim 35, further characterized in that it 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. 40.- A method for detecting RNA hybridization comprises: a) contacting a sample of RNA with an oligonucleotide probe to form a hybridized RNA; b) reacting said hybridized RNA with a metal complex capable of oxidizing a preselected base in said oligonueleotide probe in an oxidation-reduction reaction, said oligonucleotide probe having at least one of said preselected bases; c) detecting said oxidation-reduction reaction; d) determining the presence or absence of hybridized RNA from said oxidation-reduction reaction detected in the preselected pool; and e) identifying the varied base with said preselected base or the paired base with the base adjacent to the preselected base. 41. The method according to claim 40, further characterized in that the step of determining further comprises the steps of: i) measuring the reaction rate of said oxidation-reduction reaction, n) comparing said reaction rate measured eon the oxidation-reduction reaction rate of the transition metal complex with a single-stranded RNA; and then m) determining whether the velocity of the measured reaction is essentially the same as the rate of oxidation reaction-educes the transition metal complex with single-stranded RNA. 42. The method according to claim 40, further characterized in that said RNA sample is a sample of single-stranded RNA, said hybridized RNA is a duplex. 43.- The "Jo" method according to claim 40, further characterized in that said oligonucleotide probe includes from about 4 to about 100 bases. 44. The net according to claim 40, character furthermore because said preselected base is guanine. 45. The method according to claim 40, further characterized in that said pre-selected base is also. 46.- The method of compliance with the claim 40, further characterized in that said transition metal complex is selected from the group consisting of Ru (bpy) 3 + R? (Me2-bpy) 32+, Ru (M? 2-? Hen) 32+, Fe (bpy) 32+ , Fe (5-Cl-phen) 32+, 0s (5-Cl-? Hen) 32+ and Re? 2Í? Y) / i1 + 47.- The method in accordance with the claim 40, further characterized in that said reaction step comprises reacting transition metal complex with the sample of liibrated RNA under conditions sufficient to effect selective oxidation of the preselected base. 48. The method of conforming with the rei indication 40, further characterized because it comprises the step of amplifying and hybridizing RNA before the contact step. 49. The method according to claim 48, further characterized in that said step of amplifying said RNA sample is carried out by means of the reverse loop transcppt polymerization chain reaction. 50. The method according to claim 41, further characterized in that said step of determining the reaction rate of the oxidation-reduction reaction comprises measuring the cyclic voltarnogram of the reaction. 51.- The method in accordance with the claim 41, further characterized in that said comparison step comprises comparing the cyclic voltammogram of the reaction of the transition metal complex with the sample of hybridized RNA against the cyclic voltarnogram known as the reaction of the transition metal complex with single-stranded RNA. . 52. The method according to claim 40, further characterized in that said oligonucleotide probe is immobilized on a solid surface. 53. The method according to claim 52, further characterized in that said transition metal complex is immobilized on the solid surface.
5. 5. The method according to claim 40, further characterized by the fact that said identification step further comprises the steps of: i) measuring the reaction rate of said oxidation-network reaction detected, ii) comparing the measured reaction rate of each of the four different known oxidation-reduction reaction rates of the transition metal complex with an RNA having adenine, cytosine, guanine or tynin bound to the preselected base; m) determining which of said known oxidation-reduction reaction rates is essentially the same as the measured reaction rate. 55.- A method for detecting RNA hybridization comprising: a) contacting an RNA sample with an oligonucleotide probe to form a hybridized RNA; b) reacting said hybridized RNA with a transition metal complex capable of oxidizing a preselected base in a oligonucleotide probe in an oxidation-reduction reaction, said oligonucleotide probe having at least one of said preselected bases; c) detecting said oxidation-reduction reaction; d) measuring the reaction rate of said oxidation-reduction reaction; e) comparing said measured reaction rate with the rate of oxidation-reduction reaction of transition metal complex with a single-stranded RNA; and then f) determine whether the reaction rate measured by Ja is the same as the oxidation-reduction reaction rate of the metal complex of 9. transition with single-stranded RNA; and g) identify the varied base with said preselected base or base paired with the base adjacent to the preselected base. 56. The method according to claim 55, also bristling face because said preselected base is guanine. 57.- The method of conformation with claim 55, further characterized in that said preselected base is eni na. 58.- The method of compliance with the re vindication 55, further characterized in that said transition metal complex is selected from the group consisting of R? (B? Y) 32+, Ru (Me2-bpy) 32+, R? (Mß2-phen) 32+, Feíbpy) 32+, Fe (5-Cl-phen) 32+, 0s (5-01-phen) 32+ and Re? 2 (p and 1 + 59 ..- The method in accordance with the claim 55, further characterized in that said reaction step comprises contacting said transition metal complex with the RNA sample under conditions sufficient to effect the selective oxidation of said prese base. eccionada The method according to claim 55, further characterized in that it comprises the step of amplifying said RNA before the reaction step. 61.- The method according to claim 60, further characterized in that said amplification step of the hybridized RNA is carried out by chain reaction "Je polymerase, displacement amplification" chain, ligase chain reaction or amplification of nucleic acid base sequences. 62. The method according to claim 55, further characterized by the fact that said measurement step comprises measuring the cyclic voltnogram of said reaction. 63. The method according to claim 55, further characterized in that said comparison step comprises comparing the cyclic voltammogram of the reaction of the transition metal complex with the hybridized RNA sample against the known cyclic voltammogram of the reaction of the complex. Rnetal transition with RNA «Je single chain. 64. The method according to claim 55, further characterized in that said oligonucleotide probe is immobilized on a solid surface. 65.- The method according to claim 64, further characterized in that said complex transition metal is mobilized on the solid surface. 66.- The method according to claim 55, further characterized in that said step "Identification" further comprises the steps of: i) comparing the measured reaction rate of each of the 4 different known oxidation-reduction reaction rates of the metal complex «transitioning it with an RNA that has adenine, cytosine, guanine or tynine bound to the preselected base; n) Determine which of these known velocities "Reaction" The known oxidation-reduction is essentially the same as the measured reaction velocity. 67. An RNA sequencing method comprising: a) contacting an RNA sample with an oligonucleotide probe to form a hybridized RNA, said oligonucleotide probe including a presected base having a unique oxidation rate; b) reacting said hybridized RNA with a metal complex capable of oxidizing the preselected base of said oligonucleotKjo probe in an oxidation-reduction reaction, said oligonucleotide probe having a predetermined number of pre-selected bases.; c) detecting said oxidation-reduction reaction; d) determining the reaction rate of said oxidation-reduction reaction detected; and e) identify the paired base with the base prese! eccionada 68. The conforming method "J" with claim 67, further characterized in that said identification step further comprises the steps of: i) comparing the measured reaction rate of each of the four different oxidation reaction rates; known reduction of the complex < Transition metal with an RNA that also has a cytosm, guanine or tynin linked to the preselected base; n) determining which of said known oxidation-reduction reaction rates is essentially the same as the reaction rate rned? Ja. 69. The method according to claim 67, further characterized further said oligonucleotide probe further includes a preselected second base having a unique oxidation rate, wherein the oxidation rate of said second base? Reselecc? Ona. " is different from the velocity «J of oxidation of the preselected base. The method according to claim 69, further characterized in that said detection step further comprises detecting the oxidization-reduction reaction of the transition metal complex with said preselected second base; wherein the measurement step further comprises measuring the reaction rate of said oxidation-reduction reaction detected from the transition metal complex with the preselected second base and wherein said identification step further comprises identifying the paired base with the second preselected base . 71. The method according to claim 67, further characterized in that it 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 RNA sample. . 72. A method for detecting nucleic acid, said nucleic acid containing at least one preselected base, said method comprising: a) contacting said nucleic acid with a complementary nucleic acid to form a hybridized nucleic acid; b) reacting said nucleic acid with a complex of metal 90 of transition able to oxidize said L > It is pre-selected in a oxidation-reduction reaction; c) detecting said oxidation-reduction reaction; and d) determining the presence or absence of said nucleic acid from the oxidation-reduction reaction detected in the preselected base; and e) identify the paired baso with the preselected base or paired basis with the base adjacent to the preselected base. 73.- The method of compliance with the claim 72, further characterized in that said step of determining further comprises the steps of: i) measuring the speed of the reaction of said oxidation-reduction reaction, n) comparing said reaction rate with the reaction rate. n "Oxidation-reduction of the transition metal complex with a single-stranded nucleic acid; and then m) determining whether the measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded nucleic acid. The method according to claim 73, further characterized in that said step of measuring the reaction rate of the oxidation-reduction reaction comprises measuring the cyclic voltarnogram of the reaction. 75.- The method of compliance with the claim 73, further characterized in that the comparison step comprises comparing the cyclic voltarnogram of the reaction of the transition metal complex with the sample of the nucleic acid hybridized to the known cyclic voltarnogram of the reaction of the transition metal complex with the "Jo nucleic of a single chain. 76. The method according to claim 72, further characterized by said identification step comprising the steps of: (i) determining the reaction rate of said oxidation-reduction reaction detected, (n) comparing said rate of reaction measured with each of the five different known oxidation-reduction reaction rates of the transition metal complex with a nucleic acid having also cytokine, guanine, tunin or uracil attached to said preselected base; and (m) determining which of said known oxidation-reduction reaction rate is essentially the same as the measured reaction rate. 77.- The method in accordance with the claim 72, further characterized in that said nucleic acid includes from about 4 to about 100 bases. 78. The method according to claim 72, further characterized in that said preselected base is selected from the group consisting "Je guanina y ademna. 79. The method according to claim 72, further characterized in that said transition metal complex is selected from the group consisting of Ru (bpy) 32+, Ru (M? 2-bpy) 32+, Ru (Me2-) ? hen) 32+, Fe (bpy) 32+, Fe (5-Cl-? hen) 32+, 0s (5-Cl-phen) 32+ and Re? 2 (? and 1+ .80.- A method «Je conformity to the innocent claim 72, also characterized because said nucleic acid is DNA. 01.- a method of compliance with claim 72, also characterized because said "nucleus or RNA". 82. The method according to claim 72, further characterized, wherein said reaction step comprises reacting the transition metal complex with the nucleic acid under conditions sufficient to effect selective oxydation of the preselected base. 83. The method according to claim 72, further characterized in that it comprises the step of enhancing the nucleic acid before the reaction step. 84. The method of conformity is in claim 83, characterized furthermore because said step of enriching the nucleic acid is carried out by a polymerase chain reaction, amplification of "chain reaction, chain reaction" or "ligase". amplification based on nucleic acid sequence .. 85.- The method of conformity with reivi cation 72, further characterized because said nucleic acid is "nrnov? l? za" Jo on a solid surface. 86. The method according to claim 85, further characterized in that said transition metal complex is immobilized on the solid surface. 87. A method for detecting the presence or absence of a target nucleic acid in a test sample suspected to contain the same, where said target nucleic acid contains at least one base, reselece, on the , said method comprising: (a) contacting said test sample with an oligonucleotide probe that specifically binds to the target nucleic acid to form a hybridized nucleic acid; wherein said target nucleic acid eon i is at least ten or more of said pre-selected base than the probe "oligonucleotide; (b) contacting said hybridized nucleic acid with a transition metal complex that oxidizes the preselected base in an oxidation-reduction reaction; (c) detecting the presence or absence of said oxidation-reduction reaction associated with the hybridized nucleic acid; and (d) determining the presence or absence of said target nucleic acid in the test sample from said oxidation-reduction reaction detected in the activated presol base. 88. A method of compliance in claim 87 or 89, further characterized in that it comprises the step of: separating the test sample from the nucleic acid h? T > locked before the detection step. 89. A method for detecting the presence or absence of a target nucleic acid in a test sample suspected of containing it, wherein said target nucleic acid contains at least one pre-selected base, said method comprising: (a) ) contacting the test sample with an oligonucleotide probe that specifically binds the target nucleic acid to form a 10? hibphed nucLeico; wherein said oligonucleotide probe is free from said preselected base; (b) contacting said hybridized nucleic acid with a transition metal complex, and oxidizing the preselected base in an oxidation-reduction reaction; fc) detecting the presence or absence of said oxidation-eduction reaction associated with said hybridized nucleic acid; and (d) determining the presence or absence of said target nucleic acid in the test sample from the oxidation-reduction reaction "Jeteetada on the preselected base. 90. A method of conforming? Jad with claim 87 or 89, further characterized in that said target nucleic acid is longer than the oligonucleotide probe and because at least one of the preselected base is not hybridized to the probe. of oligonucleotide in said hybridized nucleic acid. 91.- A method according to claim 87 or 89, characteristic "also because said step of determination is a quantitatively determining step. 92.- A method according to claim 87 or 89, character- ized further popjue said step of determination further comprises the steps of: (i) measuring the reaction rate of said oxidation-reduction reaction detected, (ii) compare the reaction rate measured with the reaction rate of the oxidation-reduction of the transition metal complex with a single-stranded nucleic acid; and then (in) determining whether the measured reaction rate is essentially the same as the oxidation reaction rate of 1-educt ion of the ransition metal complex with single-stranded target nucleic acid. 93. The method according to claim 92, further characterized in that the measurement step of the reaction rate of the oxidation-reduction reaction comprises reducing the cyclic vol- mogram of the reaction. 94.- Flneth in accordance with the claim 9 ?, characterized by "Jernas" because the comparative step comprises comparing the cyclic voltarnogram of the reaction of the transition metal complex with the target nucleic acid sample hybridized against the known cyclic voltammogram of the reaction of the transitional complex. with single-stranded target nucleic acid. 95.- A method in accordance with the claim 87 or 89, further characterized in that said target nucleic acid includes from about 4 to about 100 bases. 96.- A method in accordance with the claim 87? 89, characterized by "Jernás" because said pre-selected base is selected from the group consisting of guanine and adenine. 97.- A method of compliance with the claim 87 or 89, further characterized in that said transition metal complex is selected from the group consisting of Ru (b? Y) 32+, Ru (Me2-t "py) 32+ Ru (Mß2 -? Hen) 32+, Fe (b? Y) 32+, Fe (5-Cl-? Hen) 32+,? S (5-Cl-phen) 32+ and ReCfe (? Y) "? +. 98.- A method according to claim 07 or 89, further ac- cording to said nucleic acid target DNAs 99.- On conformance method in claim H7 or 89, further characterized by the fact that said target nucleic acid is RNA 100. A method of confounding with the immunization 87 or 89, further characterized in that said reaction step comprises reacting the transitional etal complex with said target nucleic acid under conditions < * | ue that selective oxidation of the preset base is recited. 101. (In the method according to the rei indication 87 or 89, character- ized in addition because the "step" of amplifying the target nucleic acid before the reaction step is carried out 102 .- Fl rnetoílo in accordance with the claim 101, further characterized in that the amplification step of said sample (nucleic acid target) is carried out by polyrnerase chain reaction, chain shift amplification, ligase chain reaction or amplification based on nucleic acid sequence. 103. A method according to claim 87 or 89, further characterized in that said oligonucleotide probe is immobilized on a solid surface 104. The method according to claim 103, further characterized in that said transition metal complex. it is immobilized on said solid surface. L05. ~ A method for detecting the presence or absence of a target nucleic acid in a test sample suspected to contain the same, where the target nucleic acid contains at least one pre-selected base, said method comprising: (a) contacting said test sample with an oligonucleotide probe that specifically binds to the target nucleic acid to form a hybridized nucleic acid; wherein said oligonucleotide probe is free from said preselected base; (b) contacting said hybridized nucleic acid with a transition metal complex that oxidizes the preselected base in an oxidation-reduction reaction; (c) detecting the presence or absence of said oxidation-reduction reaction associated with the hybridized nucleic acid; and (d) determining the presence or absence of said target nucleic acid in the test sample from said oxidation-reduction reaction detected in the preselected base; wherein said preselected base in the target nucleic acid is guanine; said target nucleic acid contains cytokine and said oligonucleotide probe contains an alternating base that binds to the cytochrome in said hybridized nucleic acid; and in "Where the alternate base is selected from the group consisting of inosine and 7-deaza-gua. 106.- The method of compliance with the claim 105, further characterized in that the contacting step comprises reacting the transition metal complex with nucleic acid under conditions sufficient to effect selective oxidation of said preselected base without oxidizing the base alt er na. L07.- The method according to claim 105, further characterized in that the detection step further comprises the steps of: (i) measuring the reaction rate of said oxidation-reduction reaction detected, (n) comparing the rate of reaction measured with the rate of oxidation-reduction reaction of the transition metal complex with a single-stranded nucleic acid; and then (111) "Determine whether the measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded nucleic acid. 108. The method according to claim 105, further characterized in that the step of measuring the reaction rate of the oxidation-reduction reaction comprises measuring the cyclic voltammogram of the reaction. 109. The method according to claim 107, further characterized in that the step "Comparison compare" to compare the cyclic voltage or the reaction of the transition metal complex with the sample of target nucleic acid hybridized against the known cyclic voltarnogram of the reaction «Jel transition metal complex with single-stranded target nucleic acid. 110.- The method of compliance with the claim 105, further characterized in that said target nucleic acid L07 includes from about 4 to about 100 bases. 111.- The method in accordance with the claim 105, further characterized in that said transition metal complex is selected from the group consisting of Ru (b? Y) 3 +, R? (M? 2-bpy) 32+, R? (Me2-? Hen) 32+, Fe ( bpy) 32+, Fe (5-Cl-? hen) 32+, 0s (5-Cl-? Hen) 32+ and Re? 2 (? Y) «1+. 112. A method according to claim 105, further characterized in that said target nucleic acid is DNA. 113.- A method in accordance with the claim 105, further characterized in that said target nucleic acid is RNA. 114. The method according to claim 105, further characterized in that it comprises the step of amplifying the target nucleic acid before the reaction step. The method according to claim 114, further characterized in that the amplification step of said target nucleic acid sample is carried out by polymerase chain reaction, chain shift amplification., ligase chain reaction or amplification based on nucleic acid sequence. 116. The method according to claim 105, further characterized in that said oligonucleotide is immobilized on a solid surface. 117. The method according to claim 116, further characterized in that said transitional complex is immobilized on said solid surface. 118. A method for detecting the presence or absence of a target nucleic acid in a test sample that is suspected to contain the same, said method comprising: (a) contacting the test sample with an oligonucleotide probe. which specifically binds to the target nucleic acid to form a hybridized nucleic acid, said oligonucleotide probe having extreme terminals that are blocked for elongation by terminal transferase; (b) contacting said hybridized nucleic acid to a solution containing a preselected base in the presence of terminal transferase to produce an extension product of said target nucleic acid, with said extension product composed of the preselected base; (c) contacting said oligonucleotide probe to a transition metal complex that oxidizes the preselected base in an oxidation-reduction reaction; (d) detecting the presence or absence of said oxidation-reduction reaction; and (e) determining the presence or absence of said target nucleic acid in the test sample from said oxidation-reduction reaction detected in the preselected base. 119. A method according to claim 118, further characterized in that it comprises the step of: separating said test sample from the nucleic acid h? Br? «Ja« Jo before the detection step. 120. - A method for detecting the presence or absence of a target nucleic acid in a test sample suspected to contain the same, said method comprising: (a) providing a nucleotide capture probe, wherein said probe of capture specifically binds to the target nucleic acid; (b) contacting the test sample with said capture probe to form a nucleic acid h? bpda «Jo; (c) contacting a signal probe of said oligonucleotides to said hybridized nucleic acid, wherein said signal probe specifically binds to the target nucleic acid in the same, and wherein said signal probe contains at least one base preselected, to produce a hybridized nucleic acid sandwich; (d) p > contacting said hybridized nucleic acid sandwich with a transition metal complex that oxidizes the preselected base in an oxidation-reduction reaction; (e) detecting the presence or absence of said oxidation-reduction reaction associated with the hybridized nucleic acid sandwich; and (f) determining the presence or absence of said target nucleic acid in the test sample from the oxidation-reduction reaction detected in said preselected base. 121. A method according to claim 120, further characterized in that it comprises the step of: separating said test sample from the hybridized nucleic acid before the detection step. 122. A method according to claim 121, further characterized in that the separation step is carried out between step (b) and step (c), or between step (c) and step (d). 123. A useful electrode for the electrochemical detection of a preselected Liase in a nucleic acid, by reacting said nucleic acid with a transition metal complex capable of oxidizing said preselected base in an oxidation-reduction reaction, said electrode comprising: ( a) a conductive substrate which has a working surface formed on the same; and (b) a non-conductive polymer layer connected to said work surface, wherein said polymer layer is porous to said transition metal complex, and wherein said polymer layer binds the nucleic acid thereto. 124. An electrode according to claim 123, further characterized in that it comprises a nucleic acid bound to a polymer layer, said nucleic acid containing at least one of said pre-selected base. 125. A method for detecting a nucleic acid, said nucleic acid containing at least one preselected base, said method comprising: (a) contacting a sample containing nucleic acid to an electrode, said electrode comprising (i) a conductive substrate that has a surface «He works form« Ja on the same; and (ii) a non-conductive polymer layer connected to said work surface, wherein said polymer layer binds the nucleic acid thereto; (b) reacting the nucleic acid with a transition metal complex capable of oxidizing said base "reselecc" on an oxidation-reduction reaction, and wherein said polymer layer is porous to the transition metal complex; (c) detecting said oxidation-reduction reaction by measuring the current flow through the eLectrode; and (d) determining the presence or absence of nucleic acid from the oxidation-induced reaction detected in the preselected base. 126. A method according to claim 125, further characterized in that said step of reaction is preferred by the step of: contacting said nucleic acid with complementary nucleic acid to form a hybridized nucleic acid.