| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | October 8, 2002 |
| PATENT TITLE |
Electrochemical sensor using intercalative, redox-active moieties |
| PATENT ABSTRACT |
Compositions and methods for electrochemical detection and localization of genetic point mutations and other base-stacking perturbations within oligonucleotide duplexes adsorbed onto electrodes and their use in biosensing technologies are described. An intercalative, redox-active moiety (such as an intercalator or nucleic acid-binding protein) is adhered and/or crosslinked to immobilized DNA duplexes at different separations from an electrode and probed electrochemically in the presence or absence of a non-intercalative, redox-active moiety. Interruptions in DNA-mediated electron-transfer caused by base-stacking perturbations, such as mutations or binding of a protein to its recognition site are reflected in a difference in electrical current, charge and/or potential. -------------------------------------------------------------------------------- |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | December 29, 2000 |
| PATENT REFERENCES CITED |
Arkin et al., "Long-Range Oxidation of Guanine by Ru(III) in Duplex DNA," Chem. and Biol. 4 389-400 (1997). Beratan et al, "DNA: Insulator or Wire?" Chem. and Biol. 4 3-8 (1997). Brun et al., "Dynamics of Electron Transfer between Intercalated Polycyclic Molecules: Effect of Interspersed Bases" J. Am. Chem. Soc. 114 3656-3660 (1992). Carter et al., "Voltammetric Studies of the Interaction of Metal Chelates with DNA. 2. Tris-Chelated Complexes of Cobalt (III) and Iron (II) with 1,10-Phenanthroline and 2,2'-Bipyridine" J. Am. Chem. Soc. 111 8901-8911 (1989). Carter et al., "Electrochemical Investigations of the Interaction of Metal Chelates with DNA. 3. Electrogenerated Chemiluminescent Investigation of the Interaction of Tris(1,10-phenanthroline)ruthenium(II) with DNA," Bioconj. Chem. 1 257-263 (1990). Drmanac et al., "DNA Sequence Determination by Hybridization: A Strategy for Efficient Large-Scale Sequencing" Science 260 1649-1652 (1993). Elghanian et al., "Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles" Science 277 1078-1081 (1997). Fukui et al., "Distance Dependence of Photoinduced Electron Transfer in DNA" Angew. Chem. Int. Ed. Engl. 37 158-161 (1998). Hall et al, "Oxidative DNA Damage Though Long-Range Electron Transfer" Nature 382 731-735 (1996). Hall et al., "Sensitivity of DNA-Mediated Electron Transfer of the Intervening P-Stack: A Probe for the Integrity of the DNA Base Stack" J. Am. Chem. Soc. 119 5045-5046 (1997). Hashimoto et al., "DNA sensor: a novel electrochemical gene detection method using carbon electrode immobilized DNA probes" Chem. Abstracts p. 120(11) 246, col. 2 (1994). Hashimoto et al, "Sequence-Specific Gene Detection with a Gold Electrode Modified with DNA Probes and an Electrochemically Active Dye" Anal. Chem. 66 3830-3833 (1994). Hashimoto et al., Novel DNA Sensor for Electrochemical Gene Detection, Anal. Chim. Acta 286 219-224 (1994). Herne et al., "Characterization of DNA Probes Immobilized on Gold Surface" J. Am. Chem. Soc. 119 8916-8920 (1997). Homlin et al., "Charge Transfer through the DNA Base Stack" Angew. Chem. Int. Ed. Engl. 36 2714-2730 (1997). Jackson et al., "Recognition of DNA Base Mismatches by a Rhodium Intercalator" J. Am. Chem. Soc. 119 12986-12987 (1997). Johnston et al., "Electrochemical Measurement of the Solvent Accessibility of Nucleobases Using Electron Transfer between DNA and Metal Complexes" Am. Chem. Soc. 117 8933-8938 (1995). Kelley et al., "Electrochemistry of Methylene Blue Bound to a DNA-Modified Electrode" Bioconj. Chem. 8 31-37 (1997). Kelley et al., "Photoinduced Electron Transfer in Ethidium-Modified DNA Duplexes: Dependence on Distance and Base Stacking" J. Am. Chem. Soc. 119 9861-9870 (1997). Lamture et al., "Direct Detection of Nucleic Acid Hybridization on the Surface of a Charge Coupled Device" Nucl. Acids Res. 22 2121-2125 (1994). Leng et al., "Base specific and regioselective chemical cross-linking of daunorubicin to DNA" J. Amer. Chem. Society 118 4731-4738 (1996). Lewis et al., "Distance-Dependent Electron Transfer in DNA Hairpins" Sicence 277 673-676 (1997). Lipshutz et al., "Using Oligonucleotide Probe Arrays to Access Genetic Diversity" Biotechniques 19 442-447 (1995). Maeda et al., "DNA-Immobilized Gold Electrode for DNA-Binding Drug Sensor" Anal. Sci. 8 83-84 (1992). Maeda et al., "Mg.sup.2+ -Selective Electrode Comprising Double-Helical DNA as Receptive Entity" Chem. Lett. 1805-1808 (1994). Meade et al., "Electron Transfer through DNA: Site-Specific Modification of Duplex DNA with Ruthenium Donors and Acceptors" Angew. Chem. Int. Ed. Engl. 34 352-354 (1995). Murphy et al., "Fast Photoinduced Electron Transfer through DNA Intercalation" Proc. Natl. Acad. Sci. USA 91 5315-5319 (1994). Murphy et al., "Long-Range Photoinduced Electron Transfer through a DNA Helix", Science 262 1025-1029 (1993). Netzel, "Electron Transfer Reactions in DNA" Chem. Educ. 74 646-651 (1997). Purugganan et al., "Accelerated Electron Transfer Between Metal Complexes Mediated by DNA" Science 241 1645-1649 (1988). Rodriguez et al., "Electrochemical Studies of the Interaction of Metal Chelates with DNA: 4. Voltammetric and Electrogenerated Chemiluminescent Studies of the Interaction of Tris(2,2'-bipyridine)osmium(II) with DNA", Anal. Chem. 62 2658-2662 (1990). Wang et al, "Detection of point mutation in the p53 gene using a peptide nucleic acid biosensor" Chem. Abstracts 127(2) 135, col. 1 Abstracts 13828s (1997). Warman et al, "DNA: A Molecular Wire?" Chem. Phys. Lett. 249 319-322 (1996). |
| PATENT GOVERNMENT INTERESTS | The U.S. Government has certain rights in this invention pursuant to Grant No. GM 49216 awarded by the National Institute of Health |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of detecting one or more base-stacking perturbations in a target sequence comprising: (a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex; (b) depositing said first complex onto an electrode or an addressable multielectorde array; (c) adding an intercalative, redox-active moiety to said first complex to form a second complex; and (d) measuring an electron transfer event between said electrode or addressable multielectrode array and said intercalative, redox-active moiety as an indication for the presence or absence of said base-stacking perturbations. 2. A method according to claim 1, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof. 3. A method according to claim 1, wherein said intercalative, redox-active moiety is either noncovalently adsorbed or crosslinked to said first complex. 4. A method according to claim 1, wherein said intercalative, redox-active moiety is an intercalator. 5. A method according to claim 1, wherein said intercalative, redox-active moiety is an intercalator selected from the group consisting of phenanthridines, phenothiazines, phenazines, acridines, and anthraquinones. 6. A method according to claim 1, wherein said intercalative, redox-active moiety is daunomycin. 7. A method according to claim 1, wherein said intercalative, redox-active moiety is a part of a protein. 8. A method according to claim 1, wherein said intercalative, redox-active moiety is mut Y. 9. A method according to claim 1, wherein said electrode or addressable multielectrode array is gold. 10. A method according to claim 1, wherein said electrode or addressable multielectrode array is carbon. 11. A method according to claim 1, wherein one of said single-stranded nucleic acids is derivatized with a functionalized linker. 12. A method according to claim 11, wherein said functionalized linker is comprised of 5 to 20 .sigma. bonds. 13. A method according to claim 11, wherein said functionalized linker is thiol-terminated. 14. A method according to claim 11, wherein said functionalized linker is amine-terminated. 15. A method according to claim 1, wherein said addressable multielectrode array is comprised of a monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in length deposited onto said array, wherein each of said oligonucleotide duplexes is derivatized on one end with a functionalized linker and on the opposite end with a first single-stranded overhang of known sequence composition, and wherein one of said single-stranded nucleic acids contains a second single-stranded overhang complementary to said first single-stranded overhang on said electrode or addressable multielectrode array. 16. A method of detecting one or more base-stacking perturbations in a target sequence comprising: (a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex, wherein said nucleic acids are comprised of 12 to 25 nucleotides, and wherein one of said single-stranded nucleic acids is derivatized with a thiol-terminated linker comprised of 5 to 20 .sigma. bonds; (b) depositing said first complex onto an addressable gold multielectrode array; (c) adding daumomycin to said electrode-bound first complex to form a second complex; and (d) measuring an electron transfer event between said addressable gold multielectrode array and daunomycin as an indication for the presence or absence of said base-stacking perturbations. 17. A method according to claim 16, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof. 18. A method of detecting one or more base-stacking perturbations in a target sequence comprising: (a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex, wherein said nucleic acids are comprised of 12 to 25 nucleotides, and wherein one of said single-stranded nucleic acids is derivatized with a amine-terminated linker comprised of 5 to 20 .sigma. bonds; (b) depositing said first complex onto an addressable carbon multielectrode array; (c) adding daunomycin to said electrode-bound first complex to form a second complex; and (d) measuring an electron transfer event between said addressable carbon multielectrode array and daumonycin as an indication for the presence or absence of said base-stacking perturbations. 19. A method according to claim 18, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof. 20. A method of detecting one or more base-stacking perturbations in a target sequence comprising: (a) hybridizing a first single-stranded nucleic acid to a second single-stranded nucleic acid to form a first complex of 12 to 25 nucleotides in length, wherein said first complex contains a first single-stranded overhang of known sequence composition, and wherein said first single-stranded overhang can be the same or different; (b) depositing said first complex onto an addressable multielectrode array, wherein said addressable multielectrode array is comprised of a monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in length deposited onto said array, wherein each of said oligonucleotide duplexes is derivatized on one end with a functionalized linker and on the opposite end with a second single-stranded overhang complementary to said first single-stranded overhang; (c) adding daumomycin to said electrode-bound first complex to form a second complex; and (d) measuring an electron transfer event between said addressable multielectrode array and daunomycin as an indication for the presence or absence of said base-stacking perturbations. 21. A method according to claim 20, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof. 22. A method of detecting one or more base-stacking perturbations electrocatalytically in a target sequence comprising: (a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex; (b) depositing said first complex onto an electrode or an addressable multielectrode array to form a second complex; (c) immersing said second complex in a solution comprising an intercalative, redox-active species and a non-intercalative, redox-active species; and (d) measuring an electron transfer event as an indication for the presence or absence of said base-stacking perturbations. 23. A method according to claim 22, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof. 24. A method according to claim 22, wherein said intercalative, redox-active moiety is an intercalator. 25. A method according to claim 22, wherein said intercalative, redox-active moiety is an intercalator selected from the group consisting of phenanthridines, phenothiazines, phenazines, acridines, and anthraquinones. 26. A method according to claim 22, wherein said non-intercalative, redox-active moiety is selected from the group consisting of ferricyanide, ferrocenes, hexacyanoruthenate, and hexacyanoosmate. 27. A method according to claim 22, wherein said intercalative, redox-active moiety is a protein. 28. A method according to claim 22, wherein said intercalative, redox-active moiety is methylene blue, and wherein said non-intercalative, redox-active moiety is ferricyanide. 29. A method according to claim 22, wherein said electrode or addressable multielectrode array is gold. 30. A method according to claim 22, wherein said electrode or addressable multielectrode array is carbon. 31. A method according to claim 22, wherein one of said single-stranded nucleic acids is derivatized with a functionalized linker. 32. A method according to claim 31, wherein said functionalized linker is comprised of 5 to 20 .sigma. bonds. 33. A method according to claim 31, wherein said functionalized linker is thiol-terminated. 34. A method according to claim 31, wherein said functionalized linker is amine-terminated. 35. A method according to claim 22, wherein said addressable multielectrode array is comprised of a monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in length deposited onto said array, wherein each of said oligonucleotide duplexes are derivatized on one end with a functionalized linker and on the opposite end with a first single-stranded overhang of distinct sequence composition, and wherein one of said single-stranded nucleic acids contains a second single-stranded overhang complementary to said first single-stranded overhang on said electrode or addressable multielectrode array. 36. A method of detecting one or more point mutations electrocatalytically within the p53 gene, comprising: (a) forming a set of oligonucleotide duplexes of approximately 20 base-pairs in length corresponding to the approximately 600 base pair long region within exons 5 through 8 of the p53 gene, wherein said oligonucleotide duplexes are derivatized with a thiol-terminated linker comprised of 5 to 20 .sigma. bonds; (b) depositing said oligonucleotide duplexes onto an addressable gold multielectrode array; (c) denaturing said oligonucleotide duplexes by immersing them in aqueous solution at elevated temperatures for 1 minute and removing the complementary strands to form a single-stranded monolayer; (d) exposing said single-stranded monolayer to a first sample comprising PCR-amplified and fragmented p53 gene DNA under hybridizing conditions to form a first complex; (e) rinsing said electrode-bound first complex to remove any unhybridized material; (f) immersing said electrode-bound first complex into a dilute solution comprised of 1.0 .mu.M methylene blue and 1.0 mM ferricyanide; (g) measuring an electron transfer event as an indication for the presence or absence of said point mutations; (h) denaturing said electrode-bound first complex by immersing it in an aqueous solution at elevated temperatures for 1 minute, and regenerating said single-stranded monolayer; (i) exposing said single-stranded monolayer to a second sample containing PCR-amplified and fragmented p53 gene DNA under hybridizing conditions to form a second complex; and (k) repeating steps (e) through (h) using several sample solutions. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
3. FIELD OF THE INVENTION The present invention relates to the detection and localization of base-pair mismatches and other perturbations in base-stacking within an oligonucleotide duplex. 4. DESCRIPTION OF RELATED ART It is now well known that mutations in DNA can lead to severe consequences in metabolic functions (e.g., regulation of gene expression, modulation of protein production) which ultimately are expressed in a variety of diseases. For example, a significant number of human cancers are characterized by a single base mutation in one of the three ras genes (Bos, 1989). In order to unravel the genetic components of such diseases, it is of utmost importance to develop DNA sensors that are capable of detecting single-base mismatches rapidly and efficiently and to establish routine screening of disease-related genetic mutations based on such sensors (Skogerboe, 1993; Southern, 1996; Chee, 1996; Eng, 1997). Various methods that have been developed for the detection of differences between DNA sequences rely on hybridization events to differentiate native versus mutated sequences and are limited by the small differences in base-pairing energies caused by point mutations within extended polynucleotides (Millan, 1993; Hashimoto, 1994; Xu, 1995; Wang, 1996; Lockhart, 1996; Alivisatos, 1996; Korriyoussoufi, 1997; Elghanian, 1997; Lin, 1997; Herne, 1997). Typically, a nucleic acid hybridization assay to determine the presence of a particular nucleotide sequence (i.e. the "target sequence") in either RNA or DNA comprises a multitude of steps. First, an oligonucleotide probe having a nucleotide sequence complementary to at least a portion of the target sequence is labeled with a readily detectable atom or group. When the labeled probe is exposed to a test sample suspected of containing the target nucleotide sequence, under hybridizing conditions, the target will hybridize with the probe. The presence of the target sequence in the sample can be determined qualitatively or quantitatively in a variety of ways, usually by separating the hybridized and non-hybridized probe, and then determining the amount of labeled probe which is hybridized, either by determining the presence of label in probe hybrids or by determining the quantity of label in the non-hybridized probes. Suitable labels may provide signals detectable by luminescence, radioactivity, colorimetry, x-ray diffraction or absorption, magnetism or enzymatic activity, and may include, for example, fluorophores, chromophores, radioactive isotopes, enzymes, and ligands having specific binding partners. However, the specific labeling method chosen depends on a multitude of factors, such as ease of attachment of the label, its sensitivity and stability over time, rapid and easy detection and quantification, as well as cost and safety issues. Thus, despite the abundance of labeling techniques, the usefulness, versatility and diagnostic value of a particular system for detecting a material of interest is often limited. Some of the currently used methods of mismatch detection include single-strand conformation polymorphism (SSCP) (Thigpen, 1992; Orita, 1989), denaturing gradient gel electrophoresis (DGGE) (Finke, 1996; Wartell, 1990; Sheffield, 1989), RNase protection assays (Peltonen and Pulkkinen, 1986; Osborne, 1991), allele-specific oligonucleotides (Wu, 1989), allele-specific PCR (Finke, 1996), and the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, 1991). In the first three methods, the appearance of a new electrophoretic band is observed by polyacrylamide gel electrophoresis. SSCP detects the differences in speed of migration of single-stranded DNA sequences in polacrylamide gel electrophoresis under different conditions such as changes in pH, temperature, etc. A variation in the nucleotide base sequence of single-stranded DNA segments (due to mutation or polymorphism) may lead to a difference in spatial arrangement and thus in mobility. DGGE exploits differences in the stability of DNA segments in the presence or absence of a mutation. Introduction of a mutation into double-stranded sequences creates a mismatch at the mutated site that destabilizes the DNA duplex. Using a gel with an increasing gradient of formamide (denaturation gradient gel), the mutant and wild-type DNA can be differentiated by their altered migration distances. The basis for the RNase protection assay is that the RNase A enzyme cleaves mRNA that is not fully hybridized with its complementary strand, whereas a completely hybridized duplex is protected from RNase A digestion. The presence of a mismatch results in incomplete hybridization and thus cleavage by RNase A at the mutation site. Formation of these smaller fragments upon cleavage can be detected by polyacrylamide gel electrophoresis. Techniques based on mismatch detection are generally being used to detect point mutations in a gene or its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of tumor samples. In addition to the RNase A protection assay, there are other DNA probes that can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Smooker and Cotton, 1993; Cotton, 1988; Shenk, 1975. Other enzymatic methods include for example the use of DNA ligase which covalently joins two adjacent oligonucleotides which are hybridized on a complementary target nucleic acid, see, for example Landegren (1988). The mismatch must occur at the site of ligation. Alternatively, mismatches can also be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes (Cariello, 1988). With either riboprobes or DNA probes, the cellular mRNA or DNA which may contain a mutation can be amplified using polymerase chain reaction (PCR) prior to hybridization. Changes in DNA of the gene itself can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions. DNA sequences of the specified gene which have been amplified by use of PCR may also be screened using allele-specific oligonucleotide probes. These probes are nucleic acid oligomers, each of which is complementary to a corresponding segment of the investigated gene and may or may not contain a known mutation. The assay is performed by detecting the presence or absence of a hybridization signal for the specific sequence. In the case of allele-specific PCR, the PCR technique uses unique primers which selectively hybridize at their 3'-ends to a particular mutated sequence. If the particular mutation is not present, no amplification product is observed. In addition, restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. However, since the recognition site of restriction endonucleases ranges in general between 4 to 10 base pairs, only a small portion of the genome is monitored by any one enzyme. Another means for identifying base substitution is direct sequencing of a nucleic acid fragment. The traditional methods are based on preparing a mixture of randomly-terminated, differentially labeled DNA fragments by degradation at specific nucleotides, or by dideoxy chain termination of replicating strands (Maxam & Gilbert, 1980; Sanger, 1977). Resulting DNA fragments in the range of 1 to 500 basepairs are then separated on a gel to produce a ladder of bands wherein the adjacent samples differ in length by one nucleotide. The other method for sequencing nucleic acids is sequencing by hybridization (SBH, Drmanac, 1993). Using mismatch discriminative hybridization of short n-nucleotide oligomers (n-mers), lists of constitutent n-mers may be determined for target DNA. The DNA sequence for the target DNA may be assembled by uniquely overlapping scored oligonucleotides. Yet another approach relies on hybridization to high-density arrays of oligonucleotides to determine genetic variation. Using a two-color labeling scheme simultaneous comparison of a polymorphic target to a reference DNA or RNA can be achieved (Lipshutz, 1995; Chee, 1996; Hacia, 1996). Each of these known prior art methods for detecting base pair mismatches has limitations that affect adequate sensitivity, specificity and ease of automation of the assay. In particular, these methods are unable to detect mismatches independent of sequence composition and require carefully controlled conditions, and most methods detect multiple mismatches only. Additional shortcomings that limit these methods include high background signal, poor enzyme specificity, and/or contamination. Over the last decade, attention has also focused on DNA as a medium of charge transfer in photoinduced electron transfer reactions and its role in mutagenesis and carcinogenesis. For example, studies were performed using various octahedral metal complexes (which bind tightly to DNA by intercalation) as donors and acceptors for photoinduced electron transfer. Dppz complexes of ruthenium, osmium, cobalt, nickel, and rhenium showed tight intercalative binding and unique photophysical and electrochemical properties. No photoluminesence was observed upon irradiation of the metal complexes in aqueous solution in absence of DNA (as a result of quenching by proton transfer from the solvent), whereas in the presence of DNA excitation of the complex afforded significant, long-wavelength emission (because now the intercalated complex was protected from quenching). Studies using rhodium intercalators containing phenanthrenequinone-diimine (phi) ligands displayed tight DNA binding by preferential intercalation, some with affinities and specifities approaching DNA-binding proteins. Photoinduced electron transfer using DNA as a molecular bridge has been established in various systems. Using metal complexes intercalated into the base stack of DNA as donor and acceptor it has been proposed that the DNA .pi.-stack could promote electron transfer at long range. Additionally, the products of redox-triggered reactions of DNA bases have been detected at sites remote from intercalating oxidants (Hall, 1996; Dandliker, 1997; Hall, 1997; Arkin, 1997). For example, it has been shown that a metallointercalator can promote oxidative DNA damage through long-range hole migration from a remote site. Oligomeric DNA duplexes were prepared with a rhodium intercalator covalently attached to one end and separated spatially from 5'-GG-3' doublet sites of oxidation. Rhodium-induced photooxidation occurred specifically at the 5'-G in the 5'-GG-3' doublets and was observed up to 37 .ANG. away from the site of rhodium intercalation. In addition it was found that rhodium intercalators excited with 400 nm light, initiated the repair of a thymine dimer incorporated site-specifically in the center of a synthetic 16-mer oligonucleotide duplex. The repair mechanism was thought to proceed via oxidation of the dimer by the intraligand excited state of the rhodium complex, in which an electron deficiency (hole) is localized on the intercalated phi ligand. Like electron transfer between metallointercalators, the efficiencies of long-range oxidative processes were found to be remarkably sensitive to the coupling of the reactants into the base stack (Holmlin, 1997) and depended upon the integrity of the base stack itself (Kelley, 1997c, 1997d; Hall, 1997; Arkin, 1997) as well as on the oxidation potential. Perturbations caused by mismatches or bulges greatly diminished the yields of DNA-mediated charge transport. Other studies have reported electron transfer through DNA using nonintercalating ruthenium complexes coordinated directly to amino-modified sugars at the terminal position of oligonucleotides (Meade, 1995). In this system it was suggested that electron transfer is protein-like. In proteins, where the energetic differences in coupling depend upon .sigma.-bonded interactions, small energetic differences between systems do not cause large differences in electronic coupling. In the DNA double helix however, .pi.-stacking can contribute to electronic coupling such that small energetic differences could lead to large differences in coupling efficiency. Most recently, Lewis and coworkers measured rates of photo-oxidation of a guanine base in a DNA hairpin by an associated stilbene bound at the top of the hairpin (Lewis, 1997). By systematically varying the position of the guanine base within the hairpin and measuring the rate of electron transfer, a value for .beta., the electronic coupling parameter, could be made. Here, .beta. was found to be intermediate between that seen in proteins, with .sigma. bonded arrays, and that found for a highly coupled .pi.-bonded array. Electrochemical studies of small molecule/DNA complexes have focused primarily on solution-phase phenomena, in which DNA-induced changes in redox potentials and/or diffusion constants of organic and inorganic species have been analyzed to yield association constants (Carter, 1989, 1990; Rodriguez, 1990; Welch, 1995; Kelly, 1986; Molinier-Jumel, 1978; Berg, 1981; Plambeck, 1984). In addition, rates of guanine oxidation catalyzed by electrochemically oxidized transition-metal complexes have been used to evaluate the solvent accessibility of bases for the detection of mismatches in solution (Johnston, 1995). Electrochemical signals triggered by the association of small molecules with DNA have also been applied in the design of other novel biosensors. Toward this end, oligonucleotides have been immobilized on electrode surfaces by a variety of linkages for use in hybridization assays. These include thiols on gold (Hashimoto, 1994a, 1994b; Okahata, 1992), carbodiimide coupling of guanine residues on glassy carbon (Millan, 1993), and alkane bisphosphonate films on Al.sup.3+ -treated gold (Xu, 1994, 1995). Both direct changes in mass (measured at a quartz crystal microbalance) (Okahata, 1992) and changes in current (Hashimoto, 1994a, 1994b; Millan, 1993) or electrogenerated chemiluminesence (Xu, 1994, 1995) due to duplex-binding molecules have been used as reporters for double stranded DNA. Gold surfaces modified with thiolated polynucleotides have also been used for the detection of metal ions and DNA-binding drugs (Maeda, 1992, 1994). Other known electrochemical sensors used in an increasing number of clinical, environmental, agricultural and biotechnological applications include enzyme based biosensors. Amperometric enzyme electrodes typically require some form of electrical communication between the electrode and the active site of the redox enzyme that is reduced or oxidized by the substrate. In one type of enzyme electrode, a non-natural redox couple mediates electron transfer from the substrate-reduced enzyme to the electrode. In this scheme, the enzyme is reduced by its natural substrate at a given rate; the reduced enzyme is in turn, rapidly oxidized by a non-natural oxidizing component of a redox couple that diffuses into the enzyme, is reduced, diffuses out and eventually diffuses to an electrode where it is oxidized. Electrons from a substrate-reduced enzyme will be transferred either to the enzyme's natural re-oxidizer or, via the redox-centers of the polymer to the electrode. Only the latter process contributes to the current. Thus, it is desirable to make the latter process fast relative to the first. This can be accomplished by (a) increasing the concentration of the redox centers, or (b) assuring that these centers are fast, i.e. that they are rapidly oxidized and reduced. Most natural enzymes are not directly oxidized at electrodes without being destroyed, even if the latter are maintained at strongly oxidizing potentials. Also they are not reduced at strongly reducing potentials without being decomposed. It has, however, been shown that enzymes can be chemically modified by binding to their proteins redox couples, whereupon, if in the reduced state, they transfer electrons to an electrode. It has also been shown that when redox polycations in solution electrostatically complex polyanionic enzymes, electrons will flow in these complexes from the substrate to the enzyme, and from the enzyme through the redox polymer, to an electrode. In addition, systems have been developed where a redox-active polymer, such as poly(vinyl-pyridine), has been introduced which electrically connects the enzyme to the electrode. In this case, the polycationic redox polymer forms electrostatic complexes with the polyanionic glucose oxidase in a manner mimicking the natural attraction of some redox proteins for enzymes, e.g., cytochrome c for cytochrome c oxidase. The present invention provides a new approach for the detection of mismatches based on charge transduction through DNA. This electrochemical method is based on DNA-mediated electron transfer using intercalative, redox-active species and detects differences in electrical current or charge generated with fully base-paired duplexes versus duplexes containing a base-stacking perturbation, such as a mismatch. Carried out at an addressable multielectrode array, this method allows the processing of multiple sequences in the course of a single measurement, thus significantly improving the efficiency of screening for multiple genetic defects. Most importantly, the assay reports directly on the structural difference in base pair stacking within the hybridized duplex, rather than on a thermodynamic difference based on the condition-dependent hybridization event itself. Consequently, mismatch detection becomes independent of the sequence composition and sensors based on this approach offer fundamental advantages in both scope and sensitivity over any other existing methods. 5. SUMMARY OF THE INVENTION The present invention provides a highly sensitive and accurate method for the detection of genetic point mutations in nucleic acid sequences and its application as a biosensor. In particular, the invention relates to electrodes that are prepared by modifying their surfaces with oligonucleotide duplexes combined with an intercalative, redox-active species and their use as sensors based on an electrochemical process in which electrons are transferred between the electrode and the redox-active species. One aspect of the invention relates to methods for determining the presence of point mutations sequentially in a series of oligonucleotide duplexes using an intercalative, redox-active moiety. A preferred method comprises the steps of: (a) contacting at least one strand of a first nucleic acid molecule with a strand of a second nucleic acid molecule under hybridizing conditions, wherein one of the nucleic acid molecules is derivatized with a functionalized linker, (b) depositing this duplex onto an electrode or an addressable multielectrode array, (c) contacting the adsorbed duplex which potentially contains a base-pair mismatch with an intercalative, redox-active moiety under conditions suitable to allow complex formation, (d) measuring the amount of electrical current or charge generated as an indication of the presence of a base-pair mismatch within the adsorbed duplex, (e) treating the complex under denaturing conditions in order to separate the complex, yielding a monolayer of single-stranded oligonucleotides, and (f) rehybridizing the single-stranded oligonucleotides with another target sequence. Steps (c) through (f) can then be repeated for a sequential analysis of various oligonucleotide probes. Attenuated signals, as compared to the observed signals for fully base-paired, i.e. wild-type, sequences, will correspond to mutated sequences. In some instances, it may be desirable to crosslink the intercalative, redox-active species to the duplex and perform the assay comprised of steps (a) through (d) only. Another preferred method relates to the detection of point mutations utilizing electrocatalytic principles. More specifically, this method utilizes an electrode-bound double-stranded DNA monolayer which is immersed in a solution comprising an intercalative, redox-active species, which binds to the monolayer surface, and a non-intercalative redox-active species which remains in solution. This method comprises the steps of: (a) contacting at least one strand of a first nucleic acid molecule with a strand of a second nucleic acid molecule under hybridizing conditions, wherein one of the nucleic acid molecules is derivatized with a functionalized linker, (b) depositing this duplex which potentially contains a base-pair mismatch onto an electrode or an addressable multielectrode array, (c) immersing this complex in an aqueous solution comprising an intercalative, redox-active moiety and a non-intercalative, redox-active moiety under conditions suitable to allow complex formation, (d) measuring the amount of electrical current or charge generated as an indication of the presence of a base-pair mismatch within the adsorbed duplex, (e) treating the complex under denaturing conditions in order to separate the complex, yielding a monolayer of single-stranded oligonucleotides, and (f) rehybridizing the single-stranded oligonucleotides with another target sequence. Steps (c) through (f) can then be repeated for a sequential analysis of various oligonucleotide probes. Utilizing this method, pronounced currents and thus increased signals will be observed due to the electrocatalytic reduction of the non-intercalative, redox-active moiety by the surface-bound, redox-active moiety. Yet another aspect of the invention relates to a method of detecting the presence or absence of a protein and comprises the steps of: (a) contacting at least one strand of a first nucleic acid molecule with a strand of a second nucleic acid molecule under hybridizing conditions, wherein one of the nucleic acid molecules is derivatized with a functionalized linker and wherein the formed duplex is designed such to contain the recognition site of a nucleic acid-binding protein of choice, (b) depositing this duplex onto an electrode or an addressable multielectrode array, (c) contacting the adsorbed duplex with an intercalative, redox-active moiety under conditions suitable to allow complex formation, (d) potentially crosslinking the intercalative, redox-active moiety to the duplex, (e) immersing the complex in a first sample solution to be analyzed for the presence of the nucleic acid-binding protein, (f) measuring the amount of electrical current or charge generated as an indication of the presence or absence of the nucleic acid-binding protein in the sample solution, (g) treating the complex under appropriate conditions to remove the nucleic acid-binding protein, and (h) immersing it in a second sample solution to be analyzed for the presence of the nucleic acid-binding protein in order to separate the complex. Steps (e) through (h) can then be repeated for a sequential analysis of various sample solutions. Attenuated signals, as compared to signals measured for a reference solution without the nucleic acid-binding protein, indicate the presence of the nucleic acid-binding protein which is binding to its recognition site, thus causing a perturbation in base-stacking. The invention also relates to the nature of the redox-active moieties. The requirements of a suitable intercalative, redox-active moiety include the position of its redox potential with respect to the window within which the oligonucleotide-surface linkage is stable, as well as the synthetic feasibility of covalent attachment to the oligonucleotide. In addition, chemical and physical characteristics of the redox-active intercalator may promote its intercalation in a site-specific or a non-specific manner. In a preferred embodiment, the redox-active species is in itself an intercalator or a larger entity, such as a nucleic acid-binding protein, that contains an intercalative moiety. The nature of the non-intercalative, redox-active species for the electrocatalysis based assays depends primarily on the redox potential of the intercalative, redox-active species utilized in that assay. Yet another aspect of the invention relates to the composition and length of the oligonucleotide probe and methods of generating them. In a preferred embodiment, the probe is comprised of two nucleic acid strands of equal length. In another preferred embodiment the two nucleic acid strands are of uneven length, generating a single-stranded overhang of desired sequence composition (i.e. a "sticky end"). The length of the oligonucleotide probes range preferably from 12 to 25 nucleotides, while the single-stranded overhangs are approximately 5 to 10 nucleotides in length. These single-stranded overhangs can be used to promote site-specific adsorption of other oligonucleotides with the complementary overhang or of enzymes with the matching recognition site. The invention further relates to methods of creating a spatially addressable array of adsorbed duplexes. A preferred method comprises the steps of (a) generating duplexes of variable sequence composition that are derivatized with a functionalized linker, (b) depositing these duplexes on different sites on the multielectrode array, (c) treating the complex under denaturing conditions to yield a monolayer of single-stranded oligonucleotides, and (d) hybridizing these single-stranded oligonucleotides with a complementary target sequence. Another preferred method comprises the steps of (a) depositing 5 to 10 base-pair long oligonucleotide duplexes that are derivatized on one end with a functionalized linker and contain single-stranded overhangs (approximately 5 to 10 nucleotides long) of known sequence composition at the opposite end onto a multielectrode array, and (b) contacting these electrode-bound duplexes under hybridizing conditions with single-stranded or double-stranded oligonucleotides that contain the complementary overhang. Another aspect of the invention is directed towards the nature of the electrode, methods of depositing an oligonucleotide duplex (with or without a redox-active moiety adsorbed to it) onto an electrode, and the nature of the linkage connecting the oligonucleotide duplex to the electrode. In a preferred embodiment, the electrode is gold and the oligonucleotide is attached to the electrode by a sulfur linkage. In another preferred embodiment the electrode is carbon and the linkage is a more stable amide bond. In either case, the linker connecting the oligonucleotide to the electrode is preferably comprised of 5 to 20 .sigma. bonds. Yet another aspect of the invention relates to various methods of detection of the electrical current or charge generated by the electrode-bound duplexes combined with an intercalative, redox-active species. In a preferred embodiment, the electrical current or charge is detected using electronic methods, for example voltammetry or amperommetry, or optical methods, for example fluorescence or phosphoresence. In another preferred embodiment, the potential at which the electrical current is generated is detected by potentiommetry. 6. BRIEF DESCRIPTION OF DRAWINGS Table 1 describes the electrochemical detection of single-base mismatches based on cyclic voltammograms measured for 1.0 .mu.M daunomycin noncovalently bound to duplex-modified electrodes. FIG. 1 is a schematic diagram depicting DNA duplexes used for study of distance-dependent reduction of daunomycin. The right insert illustrates the daunomycin-guanine crosslink. The left insert shows the thiol-terminated tether which connects the duplex to the electrode surface and provides 16 .sigma.-bonds between the electrode and the base stack. FIG. 2 illustrates cyclic voltammograms of gold electrodes modified with daunomycin-crosslinked thiol-terminated duplexes (A) SH-.sup.5' ATGGATCTCATCTAC (SEQ ID NO: 1)+complement (SEQ ID NO: 25) and (B) SH-.sup.5' ATCCTACTCATGGAC (SEQ ID NO: 2)+complement (SEQ ID NO: 26), where the bold Gs represent the daunomycin crosslinking site. FIG. 3 illustrates cyclic voltammograms of gold electrodes modified with daunomycin-crosslinked thiol-terminated duplexes containing TA and CA basepairs. The oligonucleotide SH-.sup.5' ATTATATAATTGCT (SEQ ID NO: 3) was hybridized with the corresponding complements containing either a T or a C opposite from the underlined A. FIG. 4 describes the charges (Q.sub.c) measured for daunomycin at DNA-modified electrodes containing different single-base mismatches. To obtain the seven different mismatched duplexes the thiol-modified sequence, SH-.sup.5' AGTACAGTCATCGCG (SEQ ID NO: 4), was hybridized with the following seven different complements (t he mismatch is indicated in bold, and the specific basepair and the melting temperature of the duplex is given in parentheses): .sup.5' CGCGATGACTGTACT (SEQ ID NO: 5) (TA, T.sub.m =68.degree. C.), .sup.5' CGCGACGACTGTACT (SEQ ID NO: 6) (CA, T.sub.m =56.degree. C.), .sup.5' CGCGATGTCTGTACT (SEQ ID NO: 7) (TT, T.sub.m =57.degree. C.), .sup.5' CGCGATCACTGTACT (SEQ ID NO: 8) (CC, T.sub.m =56.degree. C.), .sup.5' CGCGATGGCTGTACT (SEQ ID NO: 9) (GT, T.sub.m =62.degree. C.), .sup.5' CGCGATGAATGTACT (SEQ ID NO: 10) (GA, T.sub.m =60.degree. C.), .sup.5' CGCGATGCCTGTACT (SEQ ID NO: 11) (CT, T.sub.m =58.degree. C.). FIG. 5 describes the charge obtained for DNA-modified electrodes in the presence of 1.0 .mu.M daunomycin. the identified duplexes of varying percentages of GC content (13% GC: SEQ ID NO: 12 and SEQ ID NO: 22; 53% GC: SEQ ID NO: 13 and SEQ ID NO: 23; 100% GC: SEQ ID NO: 14 and SEQ ID NO: 24) were either fully base-paired or contained a single CA mismatch. Mismatch detection measuring the electrical current or charge generated was independent of the sequence composition. FIG. 6 describes the charges (Q.sub.c) measured during the in situ detection of a CA mismatch. Electrodes were derivatized with the sequence SH-.sup.5 AGTACAGTCATCGCG (SEQ ID NO: 4), where either a C or a T was incorporated into the complement across from the underlined A. Using cyclic voltammetry, the electrochemical response of daunomycin non-covalently bound to duplex-modified electrodes was measured first for the intact TA or CA duplexes (TA vs. CA), secondly (after denaturation of the duplex) for the single stranded oligonucleotide (ss), thirdly (after rehybridization with the opposite complement) again for the duplex (CA vs. TA), and lastly (after repeating the denaturation step) again for the single-stranded oligonucleotide (ss). FIG. 7 represents a schematic illustration of electrocatalytic reduction of ferricyanide. Methylene blue (MB.sup.+) is reduced electrochemically through the DNA base stack to form leucomethylene blue (LB.sup.+). Ferricyanide is then reduced by LB.sup.+, causing the regeneration of MB.sup.+ and the observation of catalytic currents. FIG. 8 illustrates cyclic voltammograms of gold electrodes modified with thiol-terminated duplexes containing TA and CA basepairs immersed in a solution containing 1.0 .mu.M methylene blue and 1.0 mM ferricyanide. The oligonucleotide SH-.sup.5' AGTACAGTCATCGCG (SEQ ID NO: 4) was hybridized with the corresponding complements containing either a T or a C opposite from the underlined A. 7. DETAILED DESCRIPTION OF THE INVENTION The expression "amplification of polynucleotides" includes methods such as polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. These methods are well known and widely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al., 1989a (for LCR). Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from a particular gene region are preferably complementary to, and hybridize specifically to sequences in the target region or in its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly. Alternatively the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments has been described by Scharf (1986). The term "base-stacking perturbations" refers to any event that causes a perturbation in base-stacking such as, for example, a base-pair mismatch, a protein binding to its recognition site, or any other entities that form oligonucleotide adducts. The term "denaturing" refers to the process by which strands of oligonucleotide duplexes are no longer base-paired by hydrogen bonding and are separated into single-stranded molecules. Methods of denaturation are well known to those skilled in the art and include thermal denaturation and alkaline denaturation. The term "hybridized" refers to two nucleic acid strands associated with each other which may or may not be fully base-paired. The term "intercalative moieties" refers to planar aromatic or heteroaromatic moieties that are capable of partial insertion and stacking between adjacent base pairs of double-stranded oligonucleotides. These moieties may be small molecules or part of a larger entity, such as a protein. Within the context of this invention the intercalative moiety is able to generate a response or mediate a catalytic event. The term "mismatches" refers to nucleic acid bases within hybridized duplexes which are not 100% complementary. A mismatch includes any incorrect pairing between the bases of two nucleotides located on complementary strands of DNA that are not the Watson-Crick base-pairs A:T or G:C. The lack of total homology may be due to deletions, insertions, inversions, substitutions or frameshift mutations. The term "mutation" refers to a sequence rearrangement within DNA. The most common single base mutations involve substitution of one purine or pyrimidine for the other (e.g., A for G or C for T or vice versa), a type of mutation referred to as a "transition". Other less frequent mutations include "transversions" in which a purine is substituted for a pyrimidine, or vice versa, and "insertions" or "deletions", respectively, where the addition or loss of a small number (1, 2 or 3) of nucleotides arises in one strand of a DNA duplex at some stage of the replication process. Such mutations are also known as "frameshift" mutations in the case of insertion/deletion of one of two nucleotides, due to their effects on translation of the genetic code into proteins. Mutations involving larger sequence rearrangement also may occur and can be important in medical genetics, but their occurrences are relatively rare compared to the classes summarized above. The term "nucleoside" refers to a nitrogenous heterocyclic base linked to a pentose sugar, either a ribose, deoxyribose, or derivatives or analogs thereof. The term "nucleotide" relates to a phosphoric acid ester of a nucleoside comprising a nitrogenous heterocyclic base, a pentose sugar, and one or more phosphate or other backbone forming groups; it is the monomeric unit of an oligonucleotide. Nucleotide units may include the common bases such as guanine (G), adenine (A), cytosine (C), thymine (T), or derivatives thereof. The pentose sugar may be deoxyribose, ribose, or groups that substitute therefore. The terms "nucleotide analog", "modified base", "base analog", or "modified nucleoside" refer to moieties that function similarly to their naturally occurring counterparts but have been structurally modified. The terms "oligonucleotide" or "nucleotide sequence" refers to a plurality of joined nucleotide units formed in a specific sequence from naturally occurring heterocyclic bases and pentofuranosyl equivalent groups joined through phosphorodiester or other backbone forming groups. The terms "oligonucleotide analogs" or "modified oligonucleotides" refer to compositions that function similarly to natural oligonucleotides but have non-naturally occurring portions. Oligonucleotide analogs or modified oligonucleotides may have altered sugar moieties, altered bases, both altered sugars and bases or altered inter-sugar linkages, which are known for use in the art. The terms "redox-active moiety" or "redox-active species" refers to a compound that can be oxidized and reduced, i.e. which contains one or more chemical functions that accept and transfer electrons. The term "redox protein" refers to proteins that bind electrons reversibly. The simplest redox proteins, in which no prosthetic group is present, are those that use reversible formation of a disulfide bond between to cysteine residues, as in thioredoxin. Most redox proteins however use prosthetic groups, such as flavins or NAD. Many use the ability of iron or copper ions to exist in two different redox states. The present invention provides a highly sensitive and accurate method based on an electrochemical assay using intercalative, redox-active species to determine the presence and location of a single or multiple base-pair mismatches. Briefly, the system is comprised of (i) a reagent mixture comprising an electrode-bound oligonucleotide duplex to which an intercalative, redox-active moiety is associated and (ii) means for detecting and quantitating the generated electrical current or charge as an indication for the presence of a fully base-paired versus a mismatch containing duplex. The present invention is particularly useful in the diagnosis of genetic diseases that arise from point mutations. For example, many cancers can be traced to point mutations in kinases, growth factors, receptors binding proteins and/or nuclear proteins. Other diseases that arise from genetic disorders include cystic fibrosis, Bloom's syndrome, thalassemia and sickle cell disease. In addition, several specific genes associated with cancer, such as DCC, NF-1, RB, p53, erbA and the Wilm's tumor gene, as well as various oncogenes, such as abl, erbB, src, sis, ras, fos, myb and myc have already been identified and examined for specific mutations. The present invention provides methods for detecting single or multiple point mutations, wherein the oligonucleotide duplex carrying the redox-active species is adsorbed and therefore continuously exposed to an electrode whose potential oscillates between a potential sufficient to effect the reduction of said chemical moiety and a potential sufficient to effect the oxidation of the chemical moiety. This method is preferred over other methods for many reasons. Most importantly, this method allows the detection of one or more mismatches present within an oligonucleotide duplex based on a difference in electrical current measured for the mismatch-containing versus the fully base-paired duplex. Thus the method is based on the differences in base-stacking of the mismatches and is independent of the sequence composition of the hybridized duplex, as opposed to existing methods that depend on thermodynamic differences in hybridization. Furthermore, this method is nonhazardous, inexpensive, and can be used in a wide variety of applications, alone or in combination with other hybridization-dependent methods. One particular aspect of the invention relates to the method for sequential detection of mismatches within a number of nucleic acid samples which comprises the following steps. At least one strand of a nucleic acid molecule is hybridized under suitable conditions with a first nucleic acid target sequence forming a duplex which potentially contains a mismatch, and wherein one of the nucleic acids is derivatized with a functionalized linker. This duplex is then deposited onto an electrode or an addressable multielectrode array forming a monolayer. An intercalative, redox-active species (e.g., daunomycin) is noncovalently adsorbed (or crosslinked, if desired) onto this molecular lawn, and the electrical current or charge generated is measured as an indication of the presence of a base pair mismatch within the adsorbed oligonucleotide complex. Subsequent treatment of the duplexes containing the intercalative, redox-active species under denaturing conditions allows separation of the complex, yielding a single-stranded monolayer of oligonucleotides which can be rehybridized to a second oligonucleotide target sequence. The steps of duplex formation, adsorption of the intercalative, redox-active species, measurement of the electrical current or charge, and denaturation of the complex to regenerate the single-stranded oligonucleotides may be repeated as often as desired to detect in a sequential manner genetic point mutations in a variety of oligonucleotide probes. The charges passed at each of the electrodes is measured and compared to the wild-type, i.e. fully base-paired, sequences. Electrodes with attenuated signals correspond to mutated sequences, while those which exhibit no change in electrical current or charge are unmutated. Furthermore, the intensity of the signal compared to the wild-type sequence not only reports the presence of the mismatch but also describes the location of the disruption within the analyzed duplex. Another aspect of the invention relates to the method of detecting mutations utilizing electrocatalysis. Briefly, the modification of electrode surfaces with oligonucleotide duplexes provides a medium that is impenetrable by negatively charged species due to the repulsion by the high negative charge of oligonucleotides. However, electrons can be shuttled through the immobilized duplexes to redox-active intercalators localized on the solvent-exposed periphery of the monolayer, which in turn can catalytically reduce these negatively charged species. More specifically, this electrocatalytic method comprises the following steps. At least one strand of a nucleic acid molecule is hybridized under suitable conditions with a first nucleic acid target sequence forming a duplex which potentially contains a mismatch, and wherein one of the nucleic acids is derivatized with a functionalized linker. This duplex is then deposited onto an electrode or a multielectrode array forming a monolayer. The assembly is immersed into an aqueous solution containing both an intercalative, redox-active species (e.g., methylene blue) and a non-intercalative, redox-active species (e.g., ferricyanide). The electrical currents or charges corresponding to the catalytic reduction of ferricyanide mediated by methylene blue are measured for each nucleic acid-modified electrode and compared to those obtained with wild-type, i.e. fully base-paired sequences. Subsequent treatment of the duplexes under denaturing conditions allows separation of the complex, yielding a single-stranded monolayer of oligonucleotides which can be rehybridized to a second oligonucleotide target sequence. The steps of duplex formation, measurement of the catalytically enhanced electrical current or charge, and denaturation of the complex to regenerate the single-stranded oligonucleotides may be repeated as often as desired to detect in a sequential manner genetic point mutations in a variety of oligonucleotide probes. This particular method based on electrocatalysis at oligonucleotide-modified surfaces is extremely useful for systems where attenuated signals resulting from the presence of mismatches are small. The addition of a non-intercalative electron acceptor amplifies the signal intensity, and allows more accurate measurements. This approach may be particularly useful to monitor assays based on redox-active proteins which bind to the oligonucleotide-modified surface, but are not easily oxidized or reduced because the redox-active center is not intercalating. The present invention further relates to the nature of the redox-active species. These species have a reduced state in which they can accept electron(s) and an oxidized state in which they can donate electron(s). The intercalative, redox-active species that are adsorbed or covalently linked to the oligonucleotide duplex include, but are not limited to, intercalators and nucleic acid-binding proteins which contain a redox-active moiety. An intercalator useful for the specified electrochemical assays is an agent or moiety capable of partial insertion between stacked base pairs in the nucleic acid double helix. Examples of well-known intercalators include, but are not limited to, phenanthridines (e.g., ethidium), phenothiazines (e.g., methylene blue), phenazines (e.g., phenazine methosulfate), acridines (e.g., quinacrine), anthraquinones (e.g., daunomycin), and metal complexes containing intercalating ligands (e.g., phi, chrysene, dppz). Some of these intercalators may interact site-selectively with the oligonucleotide duplex. For example, the chrysene ligand is known to intercalate at the mispaired site of a duplex itself (Jackson, 1997), which can be exploited for selective localization of an intercalator. This can be in particular useful to construct a duplex monolayer which contains the intercalative, redox-active species exclusively at its periphery. In the case of redox-active nucleic acid-binding proteins, differences in DNA-mediated electron transfer between the duplex-bound protein and the electrode allow for the detection of base-pair mismatches or other base-stacking perturbations. Examples of redox-active proteins include, but are not limited to, mut Y, endonuclease III, as well as any redox-active cofactor-containing DNA-binding protein. Such proteins convert to one of an oxidized and reduced form upon reacting with a selected substrate, whereafter the operation of the electrode regenerates the other of the oxidized and reduced forms. The choice of a protein depends partially on its adsorption and binding properties to biological macromolecules, i.e. nucleic acids, and with non-biological macromolecules, whether in a homogeneous solution, or when immobilized on a surface. By changing the absorption or binding characteristics, selectivity, signal to noise ratio and signal stability in these assays can be improved. The charge of a protein affects its adsorption on surfaces, absorption in films, electrophoretic deposition on electrode surfaces, and interaction with macromolecules. It is, therefore, of importance in diagnostic and analytical systems utilizing proteins to tailor the charge of the protein so as to enhance its adsorption or its binding to the macromolecule of choice, i.e the nucleic acid. In other cases, i.e when the detection assay is used during several cycles, it is of equal importance to be able to facilitate desorption, removal, or stripping of the protein from the macromolecule. These assays require oligonucleotide duplexes that are designed such as to allow for site-specific binding of the protein of choice, which may require a single-stranded overhang. Once the protein is adsorbed onto the nucleic acid, electrons are relayed via the oligonucleotide duplex to the electrode. The nature of the non-intercalative, redox-active species used in a particular ectrocatalytic assay depends primarily on the redox potential of the intercalating, redox-active species utilized in that same assay. Examples include, but are not limited to, any neutral or negatively charged probes, for example ferricyanide/ferrocyanide, ferrocene and derivatives thereof (e.g., dimethylaminomethyl-, monocarboxylic acid-, dicarboxylic acid-), hexacyanoruthenate, and hexacyanoosmate. Yet another aspect of the invention relates to a method of detecting the presence or absence of a protein inducing base-stacking perturbations in DNA duplexes, this method comprising the following steps. At least one strand of a nucleic acid molecule is hybridized under suitable conditions with a second strand of nucleic acid molecule forming a duplex, wherein one of the nucleic acids is derivatized with a functionalized linker. This duplex is designed such to contain the recognition site of a protein of choice at a distinct site along that duplex. This duplex is then deposited onto an electrode or an addressable multielectrode array forming a monolayer and an intercalative, redox-active species is adsorbed onto this molecular lawn. In a preferred embodiment, the intercalative, redox-active species is site-specifically localized. In another preferred embodiment, the intercalative, redox-active species is crosslinked to the oligonucleotide duplex. This formed complex is then exposed to a sample solution that potentially contains the specific protein and the electrical current or charge generated is measured as an indication of the presence or absence of the protein. Subsequently, the protein is removed under appropriate conditions to regenerate the duplex containing the intercalative, redox-active moiety. The steps of duplex formation, adsorption or crosslinking of the intercalative, redox-active species, measurement of the electrical current or charge, and regeneration of the duplex containing the intercalative, redox-active moiety may be repeated as often as desired to detect in a sequential manner the presence of a specific protein in multiple sample solutions. The charges passed at each of the electrodes are measured and compared to the charges measured in a reference solution without the protein. Electrodes with attenuated signals indicate the presence of the protein in question which is binding to its recognition site, thus causing a perturbation in base-stacking. Examples of proteins that can be used for this assay include, but are not limited to, restriction enzymes, TATA-binding proteins, and base-flipping enzymes (e.g., DNA methylase). The present invention also relates to the choice of nucleic acid probes. Any nucleic acid, DNA or RNA, can be subjected to this mismatch detection method, provided that the mismatch(es) to be detected lie within the region between the attachment site of the intercalative, redox-active moiety and the electrode in order to be able to measure a difference in electrical current. The nucleic acid probes to be compared may comprise natural or synthetic sequences encoding up to the entire genome of an organism. These probes can be obtained from any source, for example, from plasmids, cloned DNA or RNA, or from natural DNA or RNA from any source, including bacteria, yeast, viruses, organelles and higher organisms such as plants and animals. The samples may be extracted from tissue material or cells, including blood cells, amniocytes, bone marrow cells, cells obtained from a biopsy specimen and the like, by a variety of techniques as described for example by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y. (1982), incorporated herein by reference. Alternatively, the sequences of choice can also be prepared by well known synthetic procedures. For standard DNA and RNA synthesis methods, see for example "Synthesis and Applications of DNA and RNA" ed. S. A. Narang, Academic Press, 1987, M. J. Gait, "Oligonucleotide Synthesis", IRL Press, Wash. D.C. U.S.A., 1984, and "Oligonucleotides and Analogues"ed. F. Eckstein, IRL Press, Wash. D.C. U.S.A., 1991, as incorporated herein by reference. Briefly, oligonucleotides and oligonucleotide analogs may be synthesized, conveniently through solid state synthesis of known methodology. In a preferred embodiment, the monomeric units are added to a growing oligonucleotide chain which is covalently immobilized to a solid support. Typically, the first nucleotide is attached to the support through a cleavable linkage prior to the initiation of synthesis. Step-wise extension of the oligonucleotide chain is normally carried out in the 3' to 5' direction. When the synthesis is complete, the polymer is cleaved from the support by hydrolyzing the linkage mentioned above and the nucleotide originally attached to the support becomes the 3' terminus of the resulting oligomer. Nucleic acid synthesizers such as the Applied Biosystems, Incorporated 380B are commercially available and their use is generally understood by persons of ordinary skill in the art as being effective in generating nearly any oligonucleotide or oligonucleotide analog of reasonable length which may be desired. Triester, phosphoramidite, or hydrogen phosphonate coupling chemistries are used with these synthesizers to provide the desired oligonucleotides or oligonucleotide analogs. In addition, the invention also relates to nucleic acid probes that are constructed with a defined sequence comprised of nucleotide and non-natural nucleotide monomers to restrict the number of binding sites of the intercalative, redox-active agent to one single site. For example, in the case of the redox-active intercalator daunomycin mixed nucleotide/non-natural nucleotide oligomers were prepared containing A-T and/or I-C basepairs and one discrete guanine binding site to which daunomycin is crosslinked. The non-natural nucleotides are constructed in a step-wise fashion to produce a mixed nucleotide/non-natural nucleotide polymer employing one of the current DNA synthesis methods well known in the art, see for example "Synthesis and Applications of DNA and RNA" ed. S. A. Narang, Academic Press, 1987, M. J. Gait, "Oligonucleotide Synthesis", IRL Press, Wash. D.C. U.S.A., 1984, and "Oligonucleotides and Analogues" ed. F. Eckstein, IRL Press, Wash. D.C. U.S.A., 1991. Methods and conditions used for contacting the oligonucleotide strands of two DNAs, two RNAs or one DNA and one RNA molecule under hybridizing conditions are widely known in the art. Suitable hybridization conditions may be routinely determined by optimization procedures well known to those skilled in the art to establish protocols for use in a laboratory. See e.g., Ausubel et al., Current Protocols in Molecular Biology, Vol. 1-2, John Wiley & Sons (1989); Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Springs Harbor Press (1989); and Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y. (1982), all of which are incorporated by reference herein. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength may be varied. Another aspect of the invention relates to a surface-modified electrode and its use in a bioelectrochemical process, in which electrons are transferred directly between an electrode and an electroactive biological material which is capable of accepting or donating one or more electrons. Such a bioelectrochemical process can be in either direction. In particular, the invention provides electrodes having their surface modified with oligonucleotide duplexes carrying an intercalative, redox-active moiety. The electrode can be of any material compatible with the surface modifier being adsorbed or bound thereon, including, but not limited to noble metals such as gold, silver, platinum, palladium, as well as carbon. The preferred material for the electrodes are gold and carbon. The oligonucleotide duplex can be adsorbed onto the electrode in any convenient way. Preferably, the process of preparing such a modified electrode comprises adsorbing the oligonucleotide duplex which is derivatized at the 5'-end with a functionalized linker chains onto the electrode surface in one monolayer to obtain a uniform lawn. These linkers include, but are not limited to, thiol- or amine-terminated chains. This process is generally understood by persons of ordinary skill in the art as and is relatively simple, reproducible and can easily be automated. Furthermore, the density and composition of the monolayer is subject to variation depending on the selected assay. Methods of detecting single base-pair mismatches using intercalative, redox-active moieties require a densely packed monolayer to prevent the adsorbed intercalative, redox-active moieties from diffusing into the lawn. The method for detecting the presence or absence of a protein requires preferably an uneven monolayer comprised of duplexes of variable length to allow the protein to bind effectively to its recognition site along the duplex. In addition, the present invention further relates to methods of creating a spatially addressable array of adsorbed duplexes. In a preferred embodiment, oligonucleotide duplexes of variable sequence composition that are derivatized at the 5'-end with a functionalized linker are deposited onto a multielectrode array. Subsequent treatment of these electrode-bound duplexes under denaturing conditions yields a monolayer of single-stranded oligonucleotides, which can then be hybridized with a complementary oligonucleotide probe that potentially contains a mismatch. In another preferred embodiment, short oligonucleotide duplexes (5 to 10 base-pairs in length) that are derivatized on one end with a functionalized linker and contain single-stranded overhangs (5 to 10 nucleotides in length) of designed sequence composition at the opposite end are deposited onto a multielectrode array to generate a spatially addressable matrix. These electrode-bound duplexes can then be hybridized with single-stranded or double-stranded oligonucleotides that contain the complementary overhang. Solid supports containing immobilized molecules have been extensively used in research, in clinical analyses and in the commercial production of foods and chemicals (see e.g., U.S. Pat. No. 5,153,166; Akashi, 1992). Immobilized nucleic acids are used in hybridization assays (Lamture, 1994) and immobilized proteins in radioimmuno or ELISA assays (see, U.S. Pat. No. 5,314,830). In addition, enzymes have been immobilized to facilitate their separation from product and to allow for their efficient and repetitive use. A number of important factors have to be considered in the development of an effective immobilization procedure. First, the procedure must minimize non-specific adsorption of molecules. Second, the procedure must maintain the functional integrity of the immobilized molecules. Third, the stability of the bond between the support and the immobilized molecule must be such to avoid leaching which would lead to reduced accuracy and sensitivity. Finally, the coupling procedure must be efficient enough to result in a support with a high capacity for the target molecules as well as be cost effective. Another aspect of the invention relates to measuring the electrical current as a function of degree of hybridization of the oligonucleotide duplex adsorbed onto the electrode. When the intercalative, redox-active species is exposed to electrochemical or chemical energy, the electrical current may be continuously detected using techniques well known in the art. These include, but are not limited electronic methods, for example voltammetry or amperommetry, or optical methods, for example fluorescence or phosphoresence. Generally, photoluminescence excitation and emission occur with electromagnetic radiation of between about 200 nanometers and about 900 nanometers in wavelength. Likewise, chemiluminescent and electrochemiluminescent emission generally occur with the emitted electromagnetic radiation being between about 200 nanometers and about 900 nanometers in wavelength. The potential at which the reduction or oxidation of the chemical moiety occurs depends upon its exact chemical structure as well as factors such as the pH of the solution and the nature of the electrode used. It is well known how to determine the optimal emission and excitation wavelengths in a photoluminescent system and the optimal potential and emission wavelength of an electrochemiluminescent and chemiluminescent system. There are many methods for quantifying the amount of luminescent species present. The rate of energy input into the system can provide a measure of the luminescent species. Suitable measurements include, for example, measurements of electric current when the luminescent species is generated electrochemically, the rate of reductant or oxidant utilization when the luminescent species is generated chemically or the absorption of electromagnetic energy in photoluminescent techniques. In addition, the luminescent species can be detected by measuring the emitted electromagnetic radiation. All of these measurements can be made either as continuous, event-based measurements, or as cumulative methods which add the signal over a long period of time. Event-based measurements may be carried out with photomultiplier tubes, photodiodes or phototransistors to produce electric currents proportional in magnitude to the incident light intensity, or by using charge couple devices. Examples of cumulative methods are the integration of event-based data, and the use of photographic film to provide cumulative data directly. The publications and other reference materials referred to herein describe the background of the invention and provide additional detail regarding its practice and are hereby incorporated by reference. For convenience, the reference materials are referenced and grouped in the appended bibliography. The present invention is further described in the following examples. These examples are for illustrative purposes only, and are not to be construed as limiting the scope of the invention as set forth in the appended claims. |
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