WO2003057901A2

WO2003057901A2 - Method for detecting nucleic acid molecules based on the relative movement of surfaces

Info

Publication number: WO2003057901A2
Application number: PCT/US2003/000268
Authority: WO
Inventors: Dennis M. Connolly; Charles D. Deboer; Michael D. Potter
Original assignee: Integrated Nano Technologies LLC
Current assignee: Integrated Nano Technologies LLC
Priority date: 2002-01-08
Filing date: 2003-01-06
Publication date: 2003-07-17
Anticipated expiration: 2004-07-08
Also published as: AU2003206401A8; AU2003206401A1; WO2003057901A3

Abstract

The present invention relates to methods for detecting a target nucleic acid molecule in a sample. The methods involve providing a device having surfaces not in contact with each other, at least one of which is moveable. A pair of oligonucleotide probes complementary to the target nucleic acid molecule are attached to the surfaces of the device. The device is then contacted with a sample, under selective hybridization conditions, to permit target molecules, if any, present in the sample, to hybridize to the probes. Finally, it is determined whether there is a decrease in the relative movement between the surfaces, thereby indicating the presence of the target molecule in the sample. Alternatively, single stranded nucleic acid molecules are attached to the surfaces to determine whether the two surfaces are closer to one another than prior to the contacting. Devices used to carry out the above methods are also disclosed.

Description

METHOD FOR DETECTING NUCLEIC ACID MOLECULES BASED ON THE RELATIVE MOVEMENT OF SURFACES

FIELD OF THE INVENTION

The present invention relates to methods and apparatus for the detection of target nucleic acid molecules based on the relative movement of surfaces in samples.

BACKGROUND OF THE INVENTION

For the analysis and testing of nucleic acid molecules, amplification of a small amount of nucleic acid molecules, isolation of the amplified nucleic acid fragments, and other procedures are necessary. The polymerase chain reaction method is widely used for the amplification of nucleic acid molecules, in which an extremely small number of nucleic acid molecules or fragments can be multiplied by several orders of magnitude to provide detectable amounts of material. On the other hand, isolation and detection of particular nucleic acid molecules in a mixture requires a nucleic acid sequencer and fragment analyzer, in which gel electrophoresis and fluorescence detection are combined. However, electrophoresis becomes very labor- intensive as the number of samples or test items increases. For this reason, a simpler method of analysis using oligonucleotide probes is becoming popular. In this method, many kinds of oligonucleotide probes are immobilized on the surface of a solid to make a probe array. When contacted with a sample, only nucleic acid molecules with specific sequences matching the oligonucleotide probes are trapped on the surface of the solid and detected. This kind of isolation and detection method, in which oligonucleotide probes are immobilized on the surface of a solid and hybridization proceeds between the oligonucleotide probes and a sample, has long been known as a blotting method in which the presence of the target nucleic acid molecule is detected by a probe immobilized on a membrane using radioactive labeling. However, immobilization of a large number of probes on a small area has the advantage that only a small amount of sample is required, and a large number of probes can be used simultaneously. There are several methods for production of such products. Probe molecules can be synthesized one base at a time by a photochemical reaction on small segments of a solid using the same photomasking techniques used in the semiconductor industry. In another method, a synthesized DNA or PCR-amplified DNA is immobilized on a small segment of the surface of a solid for each probe. A third method is to use an inkjet droplet to deposit the oligonucleotide probe onto the surface. After the oligonucleotide probes are attached to the surface, the sample containing the target nucleic acid molecule to be analyzed is passed over the oligonucleotide probes at a temperature conducive to rapid hybridization of the target nucleic acid molecule with the oligonucleotide probes. A washing solution then removes all the unhybridized, unbound nucleic acid molecules. The hybridized target nucleic acid molecule is then detected by using fluorescent or radioactive labels as additional materials. While such detection methods using fluorescence or radioactivity are well known and widely used, those methods are time consuming and expensive. Therefore, a need exists for a method which can rapidly detect nucleic acid molecules from samples. The present invention is directed to achieving these objectives.

SUMMARY OF THE INVENTION

The present invention relates to a method for detecting a target nucleic acid molecule in a sample. The method involves providing a device for detecting the presence of a target nucleic acid molecule in a sample. The device contains first and second surfaces not in contact with each other, at least one of which is moveable relative to the other. The device also contains first and second oligonucleotide probes, respectively, attached to the first and second surfaces. The first oligonucleotide probe is complementary to a first portion of the target nucleic acid molecule, and the second oligonucleotide probe is complementary to a second portion of the target nucleic acid molecule. Next, the method involves contacting the oligonucleotide probes with a sample which may have the target nucleic acid molecule, under selective hybridization conditions, to permit target nucleic acid molecules, if any, present in the sample to hybridize to the first and second oligonucleotide probes. The device is then electronically interrogated so that the first or second surface is put in motion. Finally, it is determined if the first or second surfaces are less movable relative to one another than prior to the contacting step. Decreased movement indicates the presence of the target nucleic acid molecule in the sample.

Another aspect of the present invention relates to a method for detecting a target nucleic acid molecule in a sample. The method involves providing a device for detecting the presence of a target nucleic acid molecule in a sample. The device contains first and second surfaces not in contact with each other, at least one of which is moveable relative to the other, and a single stranded nucleic acid molecule complementary to the target nucleic acid molecule and attached to the first and second surfaces. Next, the method involves contacting the device with a sample which may have the target nucleic acid molecule, under selective hybridization conditions, to permit target nucleic acid molecules, if any, present in the sample to hybridize to the single stranded nucleic acid molecule. Finally, it is determined if the two surfaces are closer to one another than prior to the contacting. Movement of the two surfaces closer together indicates that the target nucleic acid molecule is present in the sample. The present invention also relates to an apparatus for detecting a target nucleic acid molecule in a sample. The apparatus contains first and second surfaces not in contact with each other, at least one of which is moveable relative to the other. The apparatus also contains first and second oligonucleotide probes, respectively, attached to the first and second surfaces. The first oligonucleotide probe is complementary to a first portion of the target nucleic acid molecule, and the second oligonucleotide probe is complementary to a second portion of the target nucleic acid molecule. The apparatus also contains a device for measuring the amount of relative movement between the first and second surfaces.

Another aspect of the present invention relates to an apparatus for detecting a target nucleic acid molecule in a sample. The apparatus contains first and second surfaces not in contact with each other, at least one of which is moveable relative to the other. The apparatus also contains a single stranded nucleic acid molecule attached to the first and second surfaces as well as a device for measuring the amount of relative movement between the two surfaces.

The present invention provides a method for detecting nucleic acid molecules from a sample by changing the physical environment of a micro electro mechanical system ("MEMS") device. Specifically, a sample potentially containing a target nucleic acid molecule is contacted with the moveable surfaces of the MEMS device, where a signal is generated, indicating whether a target nucleic acid molecule is present in the sample. Such MEMS devices are fabricated by well-known silicon fabrication techniques developed and used daily in the microelectronics industry. Typical dimensions of MEMS devices are in the hundred nanometers to several micrometers range. The importance of MEMS technology lies both in the small size of the devices and in the use of planar processing technologies, related to those used in the fabrication of electronic integrated circuits, to simultaneously "machine" large numbers of relatively simple mechanical devices in an integrated fashion. In addition, the present invention requires minimal sample manipulation and preparation and provides an economical and rapid method of nucleic acid analysis. In addition, electronically generated results from the apparatus of the present invention can be easily saved on a computer and transmitted to others via the internet.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A-C show schematic views of the apparatus in accordance with the present invention. Figure 1 A shows a micromachine with oligonucleotide probes attached to the surfaces. Figure IB shows an enlarged view of the circled area of Figure 1 A, showing molecular detail of the oligonucleotide probe attachment to the surfaces. Figure 1C shows the apparatus of Figure 1A with target nucleic acid molecules hybridized to the oligonucleotide probes.

Figure 2 shows a schematic view of a micromachine with a single- stranded nucleic acid molecule attached to a pair of electrical conductors. When a target nucleic acid molecule is present in the sample and hybridizes to the single- stranded nucleic acid molecule, supercoiling of the coupled pair of the target nucleic acid molecule and the single stranded nucleic acid molecule causes the gold sol particle attached to the single-stranded nucleic acid molecule to be pulled into electrical contact with the pair of electrical conductors of the apparatus.

Figure 3 shows a schematic view of a field effect transistor with a single-stranded nucleic acid molecule attached. When a target nucleic acid molecule is present in the sample and hybridizes to the single-stranded nucleic acid molecule, supercoiling of the hybridized target nucleic acid molecule and the single stranded nucleic acid molecule causes the negatively charged gold sol particle attached to the single-stranded nucleic acid molecule to be pulled near the transistor, changing the conductivity of the transistor. Figure 4A shows a schematic view of a micromachine with a single- stranded nucleic acid molecule attached to a reflective cantilever. As shown in Figure 4B, when a target nucleic acid molecule is present in the sample and hybridizes to the single-stranded nucleic acid molecule, supercoiling of the coupled pair of the target nucleic acid molecule and the single stranded nucleic acid molecule causes the reflective cantilever to bend, changing the angle of reflection of a laser beam impinging on the cantilever.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for detecting a target nucleic acid molecule in a sample. The method involves providing a device for detecting the presence of a target nucleic acid molecule in a sample. The device contains first and second surfaces not in contact with each other, at least one of which is moveable relative to the other. The device also contains first and second oligonucleotide probes, respectively, attached to the first and second surfaces. The first oligonucleotide probe is complementary to a first portion of the target nucleic acid molecule, and the second oligonucleotide probe is complementary to a second portion of the target nucleic acid molecule. Next, the method involves contacting the oligonucleotide probes with a sample which may have the target nucleic acid molecule, under selective hybridization conditions to permit target nucleic acid molecules, if any, present in the sample to hybridize to the first and second oligonucleotide probes. The device is then electronically interrogated so that the first or second surface is put in motion. Finally, it is determined if the first or second surfaces are less movable relative to one another than prior to the contacting step. Decreased movement indicates the presence of the target nucleic acid molecule in the sample.

The present invention also relates to an apparatus for detecting a target nucleic acid molecule in a sample. The apparatus contains first and second surfaces not in contact with each other, at least one of which is moveable relative to the other. The apparatus also contains first and second oligonucleotide probes, respectively, attached to the first and second surfaces. The first oligonucleotide probe is complementary to a first portion of the target nucleic acid molecule, and the second oligonucleotide probe is complementary to a second portion of the target nucleic acid molecule. The apparatus also contains a device for measuring the amount of relative movement between the first and second surfaces.

Figure 1 A shows one embodiment of the present invention, where the first and second oligonucleotide probes 2 and 4, respectively are attached to a first and second electrical conductors 6 and 8, respectively, which are attached to the first and second surfaces 10 and 14, respectively. Arrows A and B indicate the relative motion of first surface 10, which is supported by center post 12 in a cantilever arrangement. The motion of surface 10 is driven by electrostatic forces generated by charging first and second electrical conductors 6 and 8 with opposite polarities. The charges are rapidly reversed by a signal generator at a frequency equal to the natural resonance frequency of the cantilevered surface 10, which is determined by the length, cross sectional area, density, the Young's modulus, and the moment of inertia of the material. Oligonucleotide probe molecules can be attached to the electrical conductors in a variety of ways. For example, as shown in Figure IB, oligonucleotide probe molecule 2 is terminated with a thiol group, also known as a mercapto group, that can be reacted with a electrical conductor surface 6 to form a dative bond (indicated by arrow C).

Figure 1C shows the apparatus of Figure 1A with target nucleic acid molecules hybridized to the oligonucleotide probe molecules. Thus, when target nucleic acid molecule 16, represented by the dotted lines, in a sample hybridizes to the oligonucleotide probes 2 and 4 attached to the moveable cantilevered surface 10 and surface 14, the resonance frequency of the cantilevered surface will be lower because target nucleic acid molecule 16, when hybridized to a pair of oligonucleotide probes 2 and 4, tethers the cantilevered surface 10 to surface 14. In the extreme, when there are multiple target nucleic acid molecules 16 hybridized to multiple pairs of oligonucleotide probes, no motion will occur, because the tethered target nucleic acid molecules act as a molecular glue to completely restrain the movement of cantilevered surface 10.

Another aspect of the present invention relates to a method for detecting a target nucleic acid molecule in a sample. The method involves providing a device for detecting the presence of a target nucleic acid molecule in a sample. The device contains first and second surfaces not in contact with each other, at least one of which is moveable relative to the other, and a single stranded nucleic acid molecule complementary to the target nucleic acid molecule and attached to the first and second surfaces. Next, the method involves contacting the device with a sample which may have the target nucleic acid molecule, under selective hybridization conditions, to permit target nucleic acid molecules, if any, present in the sample to hybridize to the single stranded nucleic acid molecule. Finally, it is determined if the two surfaces are closer to one another than prior to the contacting. Movement of the two surfaces closer together indicates that the target nucleic acid molecule is present in the sample. The present invention also relates to an apparatus for detecting a target nucleic acid molecule in a sample. The apparatus contains first and second surfaces not in contact with each other, at least one of which is moveable relative to the other. The apparatus also contains a single stranded nucleic acid molecule attached to the first and second surfaces as well as a device for measuring the amount of relative movement between the two surfaces.

Figure 2 shows one embodiment of this aspect of the present invention, where single-stranded nucleic acid molecule 106 is attached to first and second electrical conductors 102 and 104, respectively. When a target nucleic acid molecule is present in the sample and hybridizes to single-stranded nucleic acid molecule 106, represented by a heavy dotted line, supercoiling of the hybridized pair of the target nucleic acid molecule and the single stranded nucleic acid molecule causes gold sol particle 108 attached to single-stranded nucleic acid molecule 106 to be pulled into electrical contact with electrical conductors 102 and 104. The change in resistance caused by this contact is measured on electrical resistance meter 114 by means of electrical connectors 110 and 112. It is known by those skilled in the art that absolute contact of gold sol particle 108 with electrical conductors 102 and 104 is not necessary for a measurable signal, because electrical conductivity can occur by electron tunneling when separation distances approach the nanometer scale.

Figure 3 shows another embodiment of the present invention, where single-stranded nucleic acid molecule 206 is attached to field effect transistor 220. When a target nucleic acid molecule is present in the sample and hybridizes to single- stranded nucleic acid molecule 206, represented by a heavy dotted line, supercoiling of the hybridized pair of the target nucleic acid molecule and the single stranded nucleic acid molecule causes gold sol particle 208 attached to single-stranded nucleic acid molecule 206 to be pulled closer to field effect transistor 220. Because gold sol particle 206 is negatively charged, the electrical field in the conducting region of transistor 220 becomes more negative and the number of carriers increases, resulting in increased electrical current.

Figures 4A-B show yet another embodiment of the present invention, where single-stranded nucleic acid molecule 406 is attached to reflective cantilevered surface 402. Support 408 connects the cantilevered surface 402 to surface 404. When target nucleic acid molecule 410 is present in the sample and hybridizes to single- stranded nucleic acid molecule 406, supercoiling of the hybridized pair of target nucleic acid molecule 410 and single stranded nucleic acid molecule 406 causes reflective cantilevered surface 402 to bend, changing the angle of reflection of an interrogating laser beam, represented by arrows Ri and R₂, impinging on and reflecting off cantilevered surface 402.

The oligonucleotide probes and single stranded nucleic acid molecules of the present invention can be formed from natural nucleotides, chemically modified nucleotides, or nucleotide analogs, as long as they have activated hydroxyl groups compatible with the attachment chemistry. Such RNA or DNA analogs comprise, but are not limited to, 2'-O-alkyl sugar modifications, methylphosphonate, phosphorothioate, phosphorodithioate, formacetal, 3'-thioformacetal, sulfone, sulfamate, and nitroxide backbone modifications, amides, and analogs wherein the base moieties have been modified. In addition, analogs of oligomers may be polymers in which the sugar moiety has been modified or replaced by another suitable moiety, resulting in polymers which include, but are not limited to, polyvinyl backbones (Pitha et al., "Preparation and Properties of Poly (I-vinylcytosine)," Biochim. Biophvs. Acta, 204:381-8 (1970); Pitha et al., "Poly(l-vinyluracil): The Preparation and Interactions with Adenosine Derivatives," Biochim. Biophvs. Acta, 204:39-48 (1970), which are hereby incorporated by reference in their entirety), morpholino backbones (Summerton, et al., "Morpholino Antisense Oligomers: Design, Preparation, and Properties," Antisense Nucleic Acid Drug Dev., 7: 187-9 (1997), which is hereby incorporated by reference in its entirety) and peptide nucleic acid (PNA) analogs (Stein et al., "A Specificity Comparison of Four Antisense Types: Morpholino, 2'-O-methyl RNA, DNA, and Phosphorothioate DNA," J. Antisense Nucleic Acid Drug Dev., 7:151-7 (1997); Egholm et al., "Peptide Nucleic Acids (PNA)-Oligonucleotide Analogues with an Achiral Peptide Backbone," (1992);

Faruqi et al., "Peptide Nucleic Acid-Targeted Mutagenesis of a Chromosomal Gene in Mouse Cells," Proc. Natl. Acad. Sci. USA. 95:1398-403 (1998); Christensen et al., "Solid-Phase Synthesis of Peptide Nucleic Acids," J. Pept. Sci., 1:175-83 (1995); Nielsen et al., "Peptide Nucleic Acid (PNA). A DNA Mimic with a Peptide Backbone," Bioconjug. Chem., 5 :3-7 (1994), which are hereby incorporated by reference in their entirety). The oligonucleotide probes or single stranded nucleic acid molecules can contain the following exemplary modifications: pendant moieties, such as, proteins (including, for example, nucleases, toxins, antibodies, signal peptides and poly-L-lysine); intercalators (e.g., acridine and psoralen), chelators (e.g., metals, radioactive metals, boron and oxidative metals), alkylators, and other modified linkages (e.g., alpha anomeric nucleic acids). Such analogs include various combinations of the above-mentioned modifications involving linkage groups and/or structural modifications of the sugar or base for the purpose of improving RNAse- mediated destruction of the targeted RNA, binding affinity, nuclease resistance, and or target specificity.

The oligonucleotide probes of the present invention are preferably short nucleic acid molecules with base sequences from 6 to 60 base units in length, and more preferably, from 12 to 36 base units in length, and most preferably from 18 to 24 base units in length. It is well known by those skilled in the art that nucleic acid hybridization using an oligonucleotide probe of about 24 base units in length is very specific, with an insignificant chance of non-specific hybridization. Such oligonucleotides can be purchased from many commercial suppliers, or can be synthesized by those skilled in the art of molecular biology. Protocols for such syntheses are found in references such as "Oligonucleotides and Analogues, A Practical Approach", Edited by F. Eckstein, Oxford University Press, New York, New York, 1991, which is hereby incorporated by reference in its entirety. Details on methods of attaching oligonucleotide probe molecules to electrically conductive surfaces can be found in U.S. Provisional Patent Application Serial No. 60/310,937, filed on August 8, 2001, which is hereby incorporated by reference in its entirety.

Examples of materials for the first and second surfaces include glass, quartz and silicon as well as polymeric material such as plastic.

The target nucleic acid molecule whose sequence is to be determined is usually isolated from a tissue sample. If the target nucleic acid molecule is genomic, the sample may be from any tissue (except exclusively red blood cells). For example, whole blood, peripheral blood lymphocytes or PBMC, skin, hair or semen are convenient sources of clinical samples. These sources are also suitable if the target is RNA. Blood and other body fluids are also convenient sources for isolating viral nucleic acids. If the target nucleic acid molecule is mRNA, the sample is obtained from a tissue in which the mRNA is expressed. If the target nucleic acid molecule in the sample is RNA, it may be reverse transcribed to DNA, but need not be converted to DNA in the present invention.

When whole cells, viruses, or other tissue samples are being analyzed, it is typically necessary to extract the nucleic acids from the cells or viruses, prior to continuing with the various sample preparation operations. Accordingly, following sample collection, nucleic acids may be liberated from the collected cells, viral coat, etc., into a crude extract, followed by additional treatment to prepare the sample for subsequent operations, such as denaturation of contaminating (DNA binding) proteins, purification, filtration, and desalting.

Liberation of nucleic acids from the sample cells or viruses, and denaturation of DNA binding proteins may generally be performed by physical or chemical methods. For example, chemical methods generally employ lysing agents to disrupt the cells and extract the nucleic acids from the cells, followed by treatment of the extract with chaotropic salts, such as guanidinium isothiocyanate or urea to denature any contaminating and potentially interfering proteins. Generally, where chemical extraction and/or denaturation methods are used, the appropriate reagents may be incorporated within the extraction chamber, a separate accessible chamber, or externally introduced.

Alternatively, physical methods may be used to extract the nucleic acids and denature DNA binding proteins. U.S. Patent No. 5,304,487 to Wilding et al., which is hereby incorporated by reference in its entirety, discusses the use of physical protrusions within microchannels or sharp edged particles within a chamber or channel to pierce cell membranes and extract their contents. More traditional methods of cell extraction may also be used, e.g., employing a channel with restricted cross-sectional dimension which causes cell lysis when the sample is passed through the channel with sufficient flow pressure. Alternatively, cell extraction and denaturing of contaminating proteins may be carried out by applying an alternating electrical current to the sample. More specifically, the sample of cells is flowed through a microtubular array while an alternating electric current is applied across the fluid flow. A variety of other methods may be utilized within the device of the present invention to effect cell lysis/extraction, including, e.g., subjecting cells to ultrasonic agitation, or forcing cells through microgeometry apertures, thereby subjecting the cells to high shear stress resulting in rupture.

Following extraction, it is often desirable to separate the nucleic acids from other elements of the crude extract, e.g., denatured proteins, cell membrane particles, and the like. Removal of particulate matter is generally accomplished by filtration, fiocculation, or the like. Ideally, the sample is concentrated by filtration, which is more rapid and does not require special reagents. A variety of filter types may be readily incorporated into the device. Samples can be forced through filters that will allow only the cellular material to pass through, trapping whole organisms and broken cell debris. Further, where chemical denaturing methods are used, it may be desirable to desalt the sample prior to proceeding to the next step. Desalting of the sample, and isolation of the nucleic acid may generally be carried out in a single step, e.g., by binding the nucleic acids to a solid phase and washing away the contaminating salts or performing gel filtration chromatography on the sample. Suitable solid supports for nucleic acid binding include, e.g., diatomaceous earth, silica, or the like. Suitable gel exclusion media is also well known in the art and is commercially available from, e.g., Pharmacia and Sigma Chemical. This isolation and/or gel filtration/desalting maybe carried out in an additional chamber, or alternatively, the particular chromatographic media may be incorporated in a channel or fluid passage leading to a subsequent reaction chamber.

The probes are preferably 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.

Hybridization assays on substrate-bound oligonucleotide arrays involve a hybridization step and a detection step. In the hybridization step, the sample potentially 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.

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.

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°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, more preferably between 2 M and 6 M, most preferably between 4 M and 6 M, between 4 M and 10 M, and, optimally, at about 5 M. For example, a 5 M agent in 2 x SSPE (Sodium Chloride/Sodium Phosphate/EDTA solution) is suitable. Betaines and lower tetraalkyl ammonium salts are examples of suitable isostabilizing agents.

Betaine (N,N,N,-trimethylglycine; (Rees et al., Biochem.. (1993) 32:137-144), which is hereby incorporated by reference in its entirety) can eliminate the base pair composition dependence of DNA thermal stability. Unlike tetramethyla monium chloride ("TMACl"), 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.

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. Denaturing agents include formamide, formaldehyde, dimefhylsulfoxide ("DMSO"), tetraethyl acetate, urea, guanidine thiocyanate

("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, and potassium thiocyanate.

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") Al and cationic detergents such as, preferably, cetyltrimethylammonium bromide ("CTAB") and dodecyl trimethylammonium bromide ("DTAB"), 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.

Addition of small amounts of ionic detergents (such as N-lauroyl- sarkosine) to the hybridization buffers can also be useful. LiCl is preferred to NaCl. Hybridization can be at 20-65°C, usually 37°-45°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, 2nd Ed., Cold Spring Harbor, N.Y. (1989); and Berger and Kimmel, "Guide to Molecular Cloning Techniques," Methods in Enzvmology, Volume 152, Academic Press, Inc., San Diego, Calif. (1987); Young and Davis, Proc. Natl. Acad. Sci. USA, 80:1194 (1983), which are hereby incorporated by reference in their entirety.

The sample and hybridization reagents are placed in contact with the device and incubated. The nucleic acid hybridization reaction is well described in "Short Protocols in Molecular Biology", John Wiley and Sons, Inc., New York, New York, 1999, which is hereby incorporated by reference in its entirety. Contact can take place in any suitable container, for example, a dish or a cell specially designed to hold the device and to allow introduction and removal of fluids. Generally, incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20°C. and about 75°C, e.g., about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, or about 65°C. For probes longer than about 14 nucleotides, 37-45°C is preferred. For shorter probes, 55-65°C is preferred. More specific hybridization conditions can be calculated using formulae for determining the melting point of the hybridized region. Preferably, hybridization is carried out at a temperature at or between ten degrees below the melting temperature and the melting temperature. More preferred, hybridization is carried out at a temperature at or between five degrees below the melting temperature and the melting temperature. The sample is incubated with the oligonucleotide probes for a time sufficient to allow the desired level of hybridization between the target nucleic acid molecule and any complementary oligonucleotide probes. After incubation with the hybridization mixture, the device is washed with the hybridization buffer, which also can include the hybridization optimizing agent. These agents can be included in the same range of amounts as for the hybridization step, or they can be eliminated altogether. In another embodiment of this invention, after the oligonucleotide probes are contacted with a sample which may have the target nucleic acid molecule, the oligonucleotide probes and the selectively hybridized target nucleic acid molecule can be filled with a filling nucleic acid sequence, where the filling nucleic acid sequence is complementary to the target nucleic acid molecule and extends between the first and second oligonucleotide probes.

One or both oligonucleotide probes may be designed to specifically recognize a variation in the sequence at the end of the probe by being configured so that the first and second oligonucleotide probes abut one another when hybridized to the target nucleic acid molecule. After the target nucleic acid molecule binds to the probes, the target nucleic acid molecule is treated with nucleases to remove the ends of the molecule which did not bind to the probes. Ligase is then added so that if the target nucleic acid molecule is present and has hybridized to the first and second oligonucleotide probes, the ligase will ligate the probes together. The device can then be heated up to a temperature high enough to denature target nucleic acid molecule from the probes. The presence of the target nucleic acid molecule then can be detected, because the first and second surfaces are connected and move together.

In another embodiment of the present invention, a plurality of first and second probes can be used to determine concentrations of the target nucleic acid molecule. For example, a plurality of each of the first and second oligonucleotide probes can be provided, and the number of pairs of identical oligonucleotide probes where there is less relative movement between the first and second surfaces is identified to quantify the amount of the target nucleic acid molecule present in the sample. Alternatively, the device can contain a plurality of single stranded nucleic acid molecules, and the number of identical single stranded nucleic acid molecules where there is movement between the first and second surfaces is identified to quantify the amount of the target nucleic acid molecule present in the sample.

The present invention can be used for numerous applications, such as detection of pathogens. Thus, in another embodiment of the present invention, the oligonucleotide probes or single stranded nucleic acid molecules are complementary to genetic material of a pathogenic bacteria. Samples may be isolated from drinking water or food and rapidly screened for infectious organisms. This invention may also be used for DNA sequencing using hybridization techniques. Such methods are described in U.S. Patent No. 5,837,832 to Chee et al., which is hereby incorporated by reference in its entirety. The present invention may also be used for food and water testing. In recent times, there have been several large recalls of tainted meat products. The detection method of the present invention can be used for the in-process detection of pathogens in foods and the subsequent disposal of the contaminated materials. This could significantly improve food safety, prevent food borne illnesses and death, and avoid costly recalls. Oligonucleotide probes or single stranded nucleic acid molecules that are complementary to genetic material of common food borne pathogens, such as Salmonella and E. coll., could be designed for use within the food industry.

The present invention can also be used for real time detection of biological warfare agents, where the oligonucleotide probes or single stranded nucleic acid molecules are complementary to genetic material of a biowarfare agent. With the recent concerns of the use of biological weapons in a theater of war and in terrorist attacks, the device could be configured into a personal sensor for the combat soldier or into a remote sensor for advanced warnings of a biological threat. The devices, which can be used to specifically identify the agent, can be coupled with a modem to send the information to another location. Mobile devices may also include a global positioning system to provide both location and pathogen information.

The present invention may also be used to identify a virus, where the oligonucleotide probes or single stranded nucleic acid molecules are complementary to genetic material of a virus, hi addition, the present invention may be used to identify an individual, where the oligonucleotide probes or single stranded nucleic acid molecules are complementary to genetic material of a human. A series of probes of sufficient number to distinguish individuals with a high degree of reliability, are provided. Various polymorphism sites are used. Preferentially, the apparatus can determine the identity to a specificity of greater than one in 1 million, more preferred is a specificity of greater than one in one billion, even more preferred is a specificity of greater than one in ten billion.

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:

1. A method for detecting a target nucleic acid molecule in a sample comprising: providing a device for detecting the presence of a target nucleic acid molecule in a sample, said device comprising: first and second surfaces not in contact with each other, at least one of which is moveable relative to the other; and first and second oligonucleotide probes, respectively, attached to the first and second surfaces, wherein the first oligonucleotide probe is complementary to a first portion of the target nucleic acid molecule and the second oligonucleotide probe is complementary to a second portion of the target nucleic acid molecule; contacting the oligonucleotide probes with a sample which may have the target nucleic acid molecule under selective hybridization conditions, to permit target nucleic acid molecules, if any, present in the sample to hybridize to the first and second oligonucleotide probes; electronically interrogating the device so that the first or second surface is put in motion; and determining if the first or second surfaces are less movable relative to one another than prior to said contacting, wherein decreased movement indicates the presence of the target nucleic acid molecule in the sample.

2. A method according to claim 1, wherein the device further comprises a first electrical conductor attached to the first surface, a second electrical conductor attached to the second surface, and the first and second oligonucleotide probes are attached to the first and second electrical conductors, respectively.

3. A method according to claim 1 further comprising: filling the oligonucleotide probes and the selectively hybridized target nucleic acid molecule with a filling nucleic acid sequence after said contacting, wherein the filling nucleic acid sequence is complementary to the target nucleic acid molecule and extends between the first and second oligonucleotide probes.

4. A method according to claim 3 further comprising: contacting the target nucleic acid molecule with nucleases after said contacting the oligonucleotide probes with a sample.

5. A method according to claim 3, wherein the first and second oligonucleotide probes abut one another at a junction when hybridized to the target nucleic acid molecule, said method further comprising: contacting the target nucleic acid molecule with ligase after said filling; and heating the device to a temperature high enough to denature the target nucleic acid molecule from the probes.

6. A method according to claim 1, wherein the probes are complementary to genetic material of a pathogenic bacteria.

7. A method according to claim 6, wherein the pathogenic bacteria is a biowarfare agent.

8. A method according to claim 6, wherein the pathogenic bacteria is a food borne pathogen.

9. A method according to claim 1 , wherein the probes are complementary to genetic material of a virus.

10. A method according to claim 1, wherein the probes are complementary to genetic material of a human.

11. A method according to claim 1 , wherein one or both of the probes has a sequence which is complementary to a polymorphism where the nucleotide or nucleotides complementary to the polymorphism are located at an end of the probes.

12. A method according to claim 1, wherein the first and second oligonucleotide probes are attached to the first and second surfaces, respectively, with a linker.

13. A method according to claim 1 , wherein the first and second oligonucleotide probes are configured to hybridize to the target nucleic acid sequence at a temperature of 20-75°C.

14. A method according to claim 1 , wherein a plurality of each of the first and second oligonucleotide probes are attached to first and second surfaces, respectively, said method further comprising: identifying the number of pairs of first and second oligonucleotide probes in which there is less relative movement between the first and second surfaces to quantify the amount of the target nucleic acid molecule present in the sample.

15. A method according to claim 1, wherein the oligonucleotide probes are selected from the group consisting of DNA, RNA, chemically modified nucleic acid molecules, and nucleic acid analogs.

16. A method according to claim 1 , wherein said method is used for nucleic acid sequencing.

17. A method according to claim 1, wherein the first and second surfaces are each made from a material separately selected from the group consisting of glass, quartz, silicon, and polymeric material.

18. A method according to claim 1 , wherein the sample is saliva, whole blood, peripheral blood lymphocytes, skin, hair, or semen.

19. A method according to claim 1 , wherein said method is used to detect infectious agents.

20. A method for detecting a target nucleic acid molecule in a sample comprising: providing a device for detecting the presence of a target nucleic acid molecule in a sample, said device comprising: first and second surfaces not in contact with each other, at least one of which is moveable relative to the other; and a single stranded nucleic acid molecule complementary to the target molecule and attached to both the first and second surfaces; contacting the device with a sample which may have the target nucleic acid molecule under selective hybridization conditions, to permit target nucleic acid molecules, if any, present in the sample to hybridize to the single stranded nucleic acid molecule; and determining if the two surfaces are closer to one another than prior to said contacting, wherein movement of the two surfaces closer together indicates that the target nucleic acid molecule is present in the sample.

21. A method according to claim 20, wherein the device further comprises first and second electrical conductors attached to each of the first and second surfaces with the single stranded nucleic acid molecule attached to the first electrical conductor at one end and to the second electrical conductor at the other end.

22. A method according to claim 21, wherein the device further comprises: a gold sol particle attached to the single stranded nucleic acid molecule, and an electrical resistance meter connected to the first and second electrical conductors, and wherein said determining is carried out by determining if an electrical current can be carried between the first and second electrical conductors via the gold sol particle, said electrical current between the electrical conductors indicating the presence of the target nucleic acid molecule in the sample which has sequences complementary to the single stranded nucleic acid molecule.

23. A method according to claim 20, wherein the single stranded nucleic acid molecule has a sequence complementary to genetic material of a pathogenic bacteria.

24. A method according to claim 23, wherein the pathogenic bacteria is a biowarfare agent.

25. A method according to claim 23, wherein the pathogenic bacteria is a food borne pathogen.

26. A method according to claim 20, wherein the single stranded nucleic acid molecule has a sequence complementary to genetic material of a virus.

27. A method according to claim 20, wherein the single stranded nucleic acid molecule has a sequence complementary to genetic material of a human.

28. A method according to claim 20, wherein the single stranded nucleic acid molecule has a sequence which is complementary to a polymorphism.

29. A method according to claim 20, wherein the single stranded nucleic acid molecule is attached to the first and second surfaces, respectively, with a linker.

30. A method according to claim 20, wherein the single stranded nucleic acid molecule is configured to hybridize to the target nucleic acid molecule at a temperature of 20-75°C.

31. A method according to claim 20, wherein a plurality of single stranded nucleic acid molecules are attached to the first and second surfaces, said method further comprising: identifying the number of first and second surfaces in which there is movement to quantify the amount of the target nucleic acid molecule present in the sample.

32. A method according to claim 20, wherein the single stranded nucleic acid molecule is selected from the group consisting of DNA, RNA, chemically modified nucleic acid molecules, and nucleic acid analogs.

33. A method according to claim 20, wherein the first and second surfaces are each made from a material separately selected from the group consisting of glass, quartz, silicon, and polymeric material.

34. A method according to claim 20, wherein the sample is saliva, whole blood, peripheral blood lymphocytes, skin, hair, or semen.

35. A method according to claim 20, wherein said method is used to detect infectious agents.

36. A method according to claim 20, wherein said method is used for nucleic acid sequencing.

37. An apparatus for detecting a target nucleic acid molecule in a sample comprising: first and second surfaces not in contact with each other, at least one of which is moveable relative to the other; first and second oligonucleotide probes, respectively, attached to the first and second surfaces, wherein the first oligonucleotide probe is complementary to a first portion of the target nucleic acid molecule and the second oligonucleotide probe is complementary to a second portion of the target nucleic acid molecule; and a device for measuring the amount of relative movement between the first and second surfaces.

38. An apparatus according to claim 37 further comprising: a first electrical conductor attached to the first surface, a second electrical conductor attached to the second surface, wherein the first and second oligonucleotide probes are attached to the first and second electrical conductors, respectively.

39. An apparatus according to claim 37, wherein a plurality of the first and second oligonucleotide probes are attached to the first and second surfaces, respectively.

40. An apparatus according to claim 37, wherein the first and second surfaces are each made from a material separately selected from the group consisting of glass, quartz, silicon, and polymeric material.

41. An apparatus according to claim 37, wherein the first and second oligonucleotide probes are attached to each of the first and second surfaces with a linker.

42. An apparatus for detecting a target nucleic acid molecule in a sample comprising: first and second surfaces not in contact with each other, at least one of which is moveable relative to the other; a single stranded nucleic acid molecule attached to the first and second surfaces; and a device for measuring the amount of relative movement between the two surfaces.

43. An apparatus according to claim 42 further comprising: first and second electrical conductors attached to each of the first and second surfaces with the single stranded nucleic acid molecule attached to the first and second electrical conductors.

44. An apparatus according to claim 43, wherein a plurality of single stranded nucleic acid molecules is provided.

45. An apparatus according to claim 42, wherein the first and second surfaces are each made from a material separately selected from the group consisting of glass, quartz, silicon, and polymeric material.

46. An apparatus according to claim 42, wherein the single stranded nucleic acid molecule is attached to each of the first and second surfaces with a linker.