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High resolution dna detection methods and devicesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidHigh resolution dna detection methods and devices description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20050287589, High resolution dna detection methods and devices. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] DNA identification technology has numerous uses including identification of pathogenic organisms, genetic testing, and forensics. Advances have been made to allow for automated screening of thousands of sequences concurrently. Gene chip technologies exist where DNA probes are immobilized on a substrate such as a glass or silicon chip. A sample containing nucleic acid molecules is applied to the chip and the nucleic acid molecules within the sample are allowed to hybridize to the probe DNA on the chip. Fluorescence detection is typically used to identify double stranded nucleic acid molecule products. The advantage of the system is the ability to screen hundreds or thousands of sequences using automated systems. [0002] Hybridization screening with fluorescence detection is a powerful technique for detecting nucleic acid sequences. However, in order to detect target DNA molecules, the target must first be amplified by PCR to get a reliable signal. The gene chip technology also requires a system capable of detecting fluorescent or radioactive materials. Such a system is expensive to use and is not amenable to a portable system for biological sample detection and identification. Furthermore, the hybridization reactions take up to two hours. For many potential uses, such as detecting biological warfare agents, the gene chip system is simply not effective. Therefore, there is a need for a system which can rapidly detect small quantities of a target nucleic acid molecule without relying on PCR amplification. SUMMARY OF THE INVENTION [0003] The present invention provides a method for detecting a target nucleic acid molecule. A device for detecting the presence of a target nucleic acid molecule is provided having two electronic leads, where the ends of the leads are located near each other but are not in contact. One or more sets of two oligonucleotide probes are attached to the electronic leads. The oligonucleotide probes are positioned such that the probes can not come into contact with one another and such that a target nucleic acid molecule, which has two sequences complimentary to the probes can bind to both probes concurrently. A sample which may have the target nucleic acid molecule is contacted with the probes under selective hybridization conditions. If the target is present it bridges the gap between the probes. The target nucleic acid molecule may then carry current between the probes, or be used as a support to form a conductive wire between the two probes. [0004] The present invention also provides a device for detecting the presence of a target nucleic acid molecule. The device has two electronic leads, where the ends of the leads are located near each other but are not in contact. One or more sets of two oligonucleotide probes are attached to the electronic leads. The oligonucleotide probes are positioned such that the probes can not come into contact with one another and such that a target nucleic acid molecule, which has two sequences complimentary to the probes can bind to both probes concurrently. DESCRIPTION OF THE DRAWINGS [0005] FIG. 1 graphically depicts the method of the present invention. Two leads are provided each having a probe which is complimentary to sequences on a target nucleic acid molecule (FIG. 1A). A target nucleic acid molecule binds to the two probes at the complimentary sequences (FIG. 1B). The complimentary strand is filled in (FIG. 1C). Nucleases are used to remove the free ends of the target nucleic acid molecule (FIG. 1D). Current can be passed through the double stranded molecule or the target nucleic acid molecule and probes may be coated with a conductor and then tested for current flow. [0006] FIG. 2 is a variation on the method shown in FIG. 1 using a ligase method to distinguish a single base variation. The variation is identified by the asterisk. After step D, a ligase is used. Only those targets which have an exact match at the ends of the probes will ligate. After ligation, the sample is heated to remove non-ligated target molecules (FIG. 2E). The structure in FIG. 2E is stable at higher temperatures, whereas the un-ligated structure in FIG. 2D would denature under heat treatment. DETAILED DESCRIPTION OF THE INVENTION [0007] The present invention provides devices and methods for rapidly detecting the presence of nucleic acid molecules. The target nucleic acid molecule either itself, or as a support, is used to complete a electrical circuit. The presence of the target nucleic acid molecule is indicated by the ability to conduct an electrical signal through the circuit. In the case where the target nucleic acid molecule is not present, the circuit is not be completed. Thus, the target nucleic acid molecule acts as a switch. The presence of the nucleic acid molecule provides an on signal for an electrical circuit, whereas the lack of the target nucleotide is interpreted as an off signal. Due to the direct detection of the target nucleic acid molecule, the method allows for extremely sensitive detection of target molecules connect two wires. [0008] The detection device is constructed on a support. Examples of useful substrate materials include, e.g., glass, quartz and silicon as well as polymeric substrates, e.g. plastics. In the case of conductive or semi-conductive substrates, it will generally be desirable to include an insulating layer on the substrate. However, any solid support which has a non-conductive surface may be used to construct the device. The support surface need not be flat. In fact, the support may be on the walls of a chamber in a chip. [0009] Two leads are provided having ends located close together, within the spanning distance of a target nucleic acid molecule, but not contacting one another. Current can not flow effectively between the leads without the presence of a target nucleic acid molecule to bridge the two leads. Two probes specific to the target nucleic acid molecule are used. The first is attached to one lead, the second to the other lead. The two probes are specific to sequences on the target molecule which are separated by sufficient distance to span the region between the leads. Typically, the gap will by in micron or fractions of microns in length. However, as chip manufacturing has improved, it has become possible to shrink the distance between elements on a chip, requireing shorter lengths of target nucleic acid molecules. [0010] The target nucleic acid molecule is passed over the two leads. If a target molecule has a sequence complimentary to one of the probes, it can bind to that probe. Once bound to that probe, the nucleic acid molecule is tethered at that site. The sequence complimentary to the second probe can then bind to the second probe. To facilitate such a reaction, the two complimentary sequences should be chosen such that the length of the molecule in between can span the distance between the two leads and provide flexibility for the nucleic acid molecule to move easily, such that the second complimentary sequence readily binds to the second probe. [0011] In a preferred embodiment, the probes are selected to bind with the target such that they have approximately the same melting temperature. This can be done by varying the lengths of the hybridization region. A-T rich regions may have longer target sequences, whereas G-C rich regions would have shorter target sequences. [0012] Hybridization assays on substrate-bound oligonucleotide arrays involve a hybridization step and a detection step. In the hybridization step, a hybridization mixture containing the target and an isostabilizing agent, denaturing agent or renaturation accelerant is brought into contact with the probes of the array and incubated at a temperature and for a time appropriate to allow hybridization between the target and any complementary probes. Usually, unbound target molecules are then removed from the array by washing with a wash mixture that does not contain the target, such as hybridization buffer. This leaves only bound target molecules. In the detection step, the probes to which the target has hybridized are identified. In the present method the detection is carried out by detecting a completed electronic circuit. Since the nucleotide sequence of the probes at each feature is known, identifying the locations at which target has bound provides information about the particular sequences of these probes. [0013] Including a hybridization optimizing agent in the hybridization mixture significantly improves signal discrimination between perfectly matched targets and single-base mismatches. As used herein, the term "hybridization optimizing agent" refers to a composition that decreases hybridization between mismatched nucleic acid molecules, i.e., nucleic acid molecules whose sequences are not exactly complementary. [0014] An isostabilizing agent is a composition that reduces the base-pair composition dependence of DNA thermal melting transitions. More particularly, the term refers to compounds that, in proper concentration, result in a differential melting temperature of no more than about 1.degree. C. for double stranded DNA oligonucleotides composed of AT or GC, respectively. Isostabilizing agents preferably are used at a concentration between 1 M and 10 M, between 2 M and 6 M, between 4 M and 6 M, between 4 M and 10 M and, optimally, at about 5 M. For example, 5 M agent in 2.times.SSPE is suitable. Betaines and lower tetraalkyl ammonium salts are examples of isostabilizing agents. In one embodiment, the isostabilizing agent is not an alkylammonium ion. [0015] Betaine (N,N,N,-trimethylglycine; (Rees et al., Biochem., (1993) 32:137-144), which is hereby incorporated by reference) can eliminate the base pair composition dependence of DNA thermal stability. Unlike TMAC1, betaine is zwitterionic at neutral pH and does not alter the polyelectrolyte behavior of nucleic acids while it does alter the composition-dependent stability of nucleic acids. Inclusion of betaine at about 5 M can lower the average hybridization signal, but increases the discrimination between matched and mismatched probes. [0016] A denaturing agent is a compositions that lowers the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double-stranded nucleic acid or the hydration of nucleic acid molecules. Denaturing agents can be included in hybridization buffers at concentrations of about 1 M to about 6 M and, preferably, about 3 M to about 5.5 M. [0017] Denaturing agents include formamide, formaldehyde, DMSO ("dimethylsulfoxide"), tetraethyl acetate, urea, GuSCN, glycerol and chaotropic salts. As used herein, the term "chaotropic salt" refers to salts that function to disrupt van der Waal's attractions between atoms in nucleic acid molecules. Chaotropic salts include, for example, sodium trifluoroacetate, sodium tricholoroacetate, sodium perchlorate, guanidine thiocyanate ("GuSCN"), and potassium thiocyanate. [0018] A renaturation accelerant is a compound that increases the speed of renaturation of nucleic acids by at least 100-fold. They generally have relatively unstructured polymeric domains that weakly associate with nucleic acid molecules. Accelerants include heterogenous nuclear ribonucleoprotein ("hnRP") A1 and cationic detergents such as, preferably, CTAB ("cetyltrimethylammonium bromide") and DTAB ("dodecyl trimethylammonium bromide"), and, also, polylysine, spermine, spermidine, single stranded binding protein ("SSB"), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol. Renaturation accelerants can be included in hybridization mixtures at concentrations of about 1 mu M to about 10 mM and, preferably, 1 mu M to about 1 mM. The CTAB buffers work well at concentrations as low as 0.1 mM. [0019] Homologous nucleotide sequences can be detected by selectively hybridizing to each other. Selectively hybridizing is used herein to mean hybridization of DNA or RNA probes from one sequence to the "homologous" sequence under stringent or non-stringent conditions (Ausubel, et al., Eds., 1989, Current Protocols in Molecular Biology, Vol. I, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, at page 2.10.3, which is hereby incorporated by reference). Hybridization and wash conditions are also exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), which is hereby incorporated by reference. [0020] A variety of hybridization buffers are useful for the hybridization assays of the invention. Addition of small amounts of ionic detergents (such as N-lauroyl-sarkosine) are useful. LiCl is preferred to NaCl. Hybridization can be at 20.degree.-65.degree. C., usually 37.degree. C. to 45.degree. C. for probes of about 14 nucleotides. Additional examples of hybridization conditions are provided in several sources, including: Sambrook et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; and Berger and Kimmel, "Guide to Molecular Cloning Techniques," Methods in Enzymology, (1987), Volume 152, Academic Press, Inc., San Diego, Calif.; Young and Davis, Proc. Natl. Acad. Sci. USA, 80:1194 (1983), which are hereby incorporated by reference. Continue reading about High resolution dna detection methods and devices... Full patent description for High resolution dna detection methods and devices Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this High resolution dna detection methods and devices patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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