WO2003057901A2 - Procede permettant de detecter des molecules d'acide nucleique en fonction du mouvement relatif de surfaces - Google Patents

Procede permettant de detecter des molecules d'acide nucleique en fonction du mouvement relatif de surfaces Download PDF

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WO2003057901A2
WO2003057901A2 PCT/US2003/000268 US0300268W WO03057901A2 WO 2003057901 A2 WO2003057901 A2 WO 2003057901A2 US 0300268 W US0300268 W US 0300268W WO 03057901 A2 WO03057901 A2 WO 03057901A2
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nucleic acid
acid molecule
target nucleic
sample
attached
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PCT/US2003/000268
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WO2003057901A3 (fr
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Dennis M. Connolly
Charles D. Deboer
Michael D. Potter
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Integrated Nano-Technologies, Llc
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Publication of WO2003057901A3 publication Critical patent/WO2003057901A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
    • G01N11/10—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by moving a body within the material
    • G01N11/16—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by moving a body within the material by measuring damping effect upon oscillatory body
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2203/00—Investigating strength properties of solid materials by application of mechanical stress
    • G01N2203/0014—Type of force applied
    • G01N2203/0025—Shearing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2203/00—Investigating strength properties of solid materials by application of mechanical stress
    • G01N2203/02—Details not specific for a particular testing method
    • G01N2203/06—Indicating or recording means; Sensing means
    • G01N2203/0617—Electrical or magnetic indicating, recording or sensing means
    • G01N2203/0623—Electrical or magnetic indicating, recording or sensing means using piezoelectric gauges

Definitions

  • 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.
  • nucleic acid molecules 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.
  • 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.
  • electrophoresis becomes very labor- intensive as the number of samples or test items increases.
  • oligonucleotide probes are immobilized on the surface of a solid to make a probe array.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the second oligonucleotide probe is complementary to a second portion of the target nucleic acid molecule.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the present invention requires minimal sample manipulation and preparation and provides an economical and rapid method of nucleic acid analysis.
  • electronically generated results from the apparatus of the present invention can be easily saved on a computer and transmitted to others via the internet.
  • 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.
  • Figure 3 shows a schematic view of a field effect transistor with a single-stranded nucleic acid molecule attached.
  • 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.
  • 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
  • the second oligonucleotide probe is complementary to a second portion of the target nucleic acid molecule.
  • 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
  • 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).
  • a thiol group also known as a mercapto group
  • Figure 1C shows the apparatus of Figure 1A with target nucleic acid molecules hybridized to the oligonucleotide probe molecules.
  • target nucleic acid molecule 16 represented by the dotted lines
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 R 2 , impinging on and reflecting off cantilevered surface 402.
  • 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.
  • 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.
  • 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).
  • 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 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.
  • 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.
  • nucleic acids 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.
  • 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.
  • chaotropic salts such as guanidinium isothiocyanate or urea
  • the appropriate reagents may be incorporated within the extraction chamber, a separate accessible chamber, or externally introduced.
  • 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.
  • nucleic acids 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.
  • 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.
  • 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.
  • a 5 M agent in 2 x SSPE sodium Chloride/Sodium Phosphate/EDTA solution
  • 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.
  • TMACl tetramethyla monium chloride
  • 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
  • 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.
  • 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.
  • ionic detergents such as N-lauroyl- sarkosine
  • 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.
  • incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20°C.
  • 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.
  • 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.
  • 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.
  • 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.
  • a plurality of first and second probes can be used to determine concentrations of the target nucleic acid molecule.
  • 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.
  • 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.
  • 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.
  • 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.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés permettant de détecter une molécule d'acide nucléique cible dans un échantillon. Ces procédés comprennent les étapes consistant à : fournir un dispositif dont les surfaces ne sont pas en contact les unes avec les autres et dont au moins une surface est mobile ; fixer une paire de sondes d'oligonucléotides complémentaires à la molécule d'acide nucléique cible, aux surfaces du dispositif ; puis mettre le dispositif en contact avec un échantillon, à des conditions d'hybridation sélective, de façon à permettre l'hybridation des éventuelles molécules cibles avec les sondes ; et finalement à déterminer si le mouvement relatif des surfaces décroît, ce qui indique la présence de la molécule cible dans l'échantillon. Autre possibilité, des molécules d'acide nucléique simple brin sont fixées aux surfaces pour déterminer si les deux surfaces sont plus proches après l'étape de mise en contact. L'invention concerne également des dispositifs utilisés pour exécuter lesdits procédés.
PCT/US2003/000268 2002-01-08 2003-01-06 Procede permettant de detecter des molecules d'acide nucleique en fonction du mouvement relatif de surfaces WO2003057901A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003206401A AU2003206401A1 (en) 2002-01-08 2003-01-06 Method for detecting nucleic acid molecules based on the relative movement of surfaces

Applications Claiming Priority (2)

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US34647602P 2002-01-08 2002-01-08
US60/346,476 2002-01-08

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WO2003057901A2 true WO2003057901A2 (fr) 2003-07-17
WO2003057901A3 WO2003057901A3 (fr) 2004-07-22

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WO2005038459A3 (fr) * 2003-08-06 2005-09-15 Bridger Technologies Inc Element a liaison pour detection de substance cible

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US6406845B1 (en) * 1997-05-05 2002-06-18 Trustees Of Tuft College Fiber optic biosensor for selectively detecting oligonucleotide species in a mixed fluid sample
US6458526B1 (en) * 2000-01-28 2002-10-01 Agilent Technologies, Inc. Method and apparatus to inhibit bubble formation in a fluid

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US4847193A (en) * 1987-06-18 1989-07-11 Gene-Trak Systems Signal amplification in an assay employing a piezoelectric oscillator
US5470710A (en) * 1993-10-22 1995-11-28 University Of Utah Automated hybridization/imaging device for fluorescent multiplex DNA sequencing
US6406845B1 (en) * 1997-05-05 2002-06-18 Trustees Of Tuft College Fiber optic biosensor for selectively detecting oligonucleotide species in a mixed fluid sample
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005038459A3 (fr) * 2003-08-06 2005-09-15 Bridger Technologies Inc Element a liaison pour detection de substance cible
JP2007501400A (ja) * 2003-08-06 2007-01-25 ブリッジャー テクノロジーズ，インク． 目標物質検出用の架橋型要素部品
AU2004281449B2 (en) * 2003-08-06 2010-12-16 Bridger Technologies, Inc. Bridged element for detection of a target substance
EP2365324A3 (fr) * 2003-08-06 2011-11-16 Bridger Technologies, Inc. Élément ponté pour la détection d'une substance cible
EP2365323A3 (fr) * 2003-08-06 2011-11-16 Bridger Technologies, Inc. Élément ponté pour la détection d'une substance cible
EP2388576A1 (fr) * 2003-08-06 2011-11-23 Bridger Technologies, Inc. Élément ponté pour la détection d'une substance cible
US8078408B2 (en) 2003-08-06 2011-12-13 The Procter & Gamble Company Bridged element for detection of a target substance
KR101214179B1 (ko) * 2003-08-06 2013-01-10 더 프록터 앤드 갬블 컴퍼니 표적 물질 검출용 가교 부재
US9857366B2 (en) 2003-08-06 2018-01-02 Bridger Technologies, Inc. Bridged element for detection of a target substance

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WO2003057901A3 (fr) 2004-07-22
AU2003206401A8 (en) 2003-07-24
AU2003206401A1 (en) 2003-07-24

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