[go: up one dir, main page]

WO2010011901A1 - Détection de molécules duplex arn-adn - Google Patents

Détection de molécules duplex arn-adn Download PDF

Info

Publication number
WO2010011901A1
WO2010011901A1 PCT/US2009/051657 US2009051657W WO2010011901A1 WO 2010011901 A1 WO2010011901 A1 WO 2010011901A1 US 2009051657 W US2009051657 W US 2009051657W WO 2010011901 A1 WO2010011901 A1 WO 2010011901A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
dna
molecule
antibody
dna duplex
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2009/051657
Other languages
English (en)
Inventor
Joe Hernandez
Venkatakrishna Shyamala
Colette Cote
Nabarun Chakraborty
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Innovative Biosensors Inc
Original Assignee
Innovative Biosensors Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Innovative Biosensors Inc filed Critical Innovative Biosensors Inc
Publication of WO2010011901A1 publication Critical patent/WO2010011901A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • Nucleic acid detection assays arc useful is in many fields. Such assays are valuable in diagnosing human and animal maladies and are also valuable in research and commercial activities.
  • Figure 1 is a schematic illustration showing example aspects of the described methods for detecting a RNA-DNA duplex in a sample.
  • Figure 2 is a graph showing results of relative light units (RLU) over time of an example duplex CANARY® protocol.
  • a sample refers to any composition or solution comprising a RNA-DNA duplex, or in need of testing to determine whether it comprises a RNA-DNA duplex.
  • a RNA- DNA duplex can be formed in a sample comprising a DNA molecule or molecules by contacting the DNA with a test RNA molecule or molecules that selectively hybridizes at stringent conditions to the DNA molecule.
  • a RNA-DNA duplex can be formed in a sample comprising a RNA molecule or molecules by contacting the RNA with a test DNA molecule or molecules that selectively hybridizes at stringent conditions to the RNA molecule.
  • a plurality of test RNA or DNA molecules can be hybridized to one or more target DNA or RNA molecule to form a DNA-RNA duplex with multiple DNA or RNA molecules and/or multiple DNA-RNA duplexes wherein the DNA-RNA duplexes include different DNA or RNA molecules.
  • the hybridizing portion of the hybridizing nucleic acids can be at least 10 (e.g., 20, 25, 30, 50, 100, 1000, or 2000) nucleotides in length.
  • the hybridizing portion of the hybridizing nucleic acid is 10, 30 or 2000 nucleotides in length.
  • the hybridizing portion of the a test RNA or DNA molecule can be at least 80%, e.g., at least 95%, or at least 98%, identical to the sequence of a portion or all of a nucleic acid encoding a target RNA or target DNA molecule.
  • Hybridization of a test nucleic acid to a target nucleic acid can be performed under stringent conditions.
  • Nucleic acid duplex stability can be expressed as the melting temperature or Tm, which is the temperature at which a test nucleic acid dissociates from a target nucleic acid. This melting temperature can be used to define stringency conditions.
  • a target sequence is substantially identical to a test sequence, then the lowest temperature at which only homologous hybridization with a particular concentration of salt (e.g., SSC or SSPE) can be determined. Assuming that a 1% mismatch results in a I 0 C decrease in the Tm, the temperature of the final wash in the final hybridization reaction is reduced accordingly (for example, if test sequences having > 95% identity with the target are sought, the final wash temperature is decreased by 5 0 C).
  • the parameters of salt concentration and temperature can be varied. Additional guidance regarding such sample conditions is readily available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubcl ct al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).
  • RNA-DNA duplex varying concentrations of RNA with ssDNA in transcription buffer from Ambion® mMessage Machine Kit [0.1X final] (Applied Biosystems, Austin, TX), and 0.65U RNase Inhibitor (NEB #AM2682) (Applied Biosystems, Austin, TX) can be mixed.
  • the reagent mixture can be incubated at 95 0 C 2 minutes to denature the nucleic acid, and then at 65 0 C 30 minutes followed by 5O 0 C 30 minutes for annealing nucleic acid.
  • the reaction mix can be diluted in assay buffer for use in the CANARY® (Innovative Biosensors, Rockville, MD) assay.
  • mixes can be prepared with RNA alone, ssDNA alone and dsDNA.
  • a target DNA or RNA molecule present in a sample can be selectively hybridized with a test RNA or DNA molecule.
  • the test RNA or DNA molecule is complementary to a portion of the target RNA or DNA molecule and hybridizes along the full length of the target RNA or DNA molecule.
  • the test RNA or DNA molecule is complementary to a portion of the target RNA or DNA molecule and hybridizes along a portion of the target RNA or DNA molecule.
  • the sample comprising a RNA-DNA duplex can be contacted with a sensor cell expressing on its sensory cell surface a RNA-DNA binding antibody or antibody fragment.
  • the antibody or antibody fragment binds with the RNA-DNA duplex in the sample and the binding of the sensor cell with the RNA-DNA duplex is detectable.
  • the detected binding indicates the presence of a RNA-DNA duplex in the sample.
  • Sensor cells, and methods and devices for using the same, are described in U.S. Patent 6,087,114, 6,248,542, 5,541,309, 5,360,728, 5,139,973 and 5,126,276, which are incorporated herein in their entirety.
  • Figure 1 is a schematic illustration showing example aspects 100 of the described methods for detecting a RNA-DNA duplex in a sample.
  • a target DNA or RNA 102 is provided. If the target to be detected is DNA, a test RNA molecule is contacted with the DNA target to form a RNA-DNA duplex sample. If the target to be detected is RNA, a test DNA molecule is contacted with the RNA target to from a RNA-DNA duplex sample.
  • the test RNA or DNA molecule can be complementary to the target DNA or RNA molecule along both the full length (104 and 110) of the test and target molecule. In other examples, the test RNA or DNA molecule can be complementary to portions of the target DNA or RNA molecule (106 and 108). In this latter case, the test RNA or DNA molecule can be shorter than the target DNA or RNA molecule.
  • a RNA-DNA duplex 108 or 1 10 can be contacted with a sensor cell 116.
  • the sensor cell can comprise a RNA-DNA duplex binding antibody or fragment 118 on its surface.
  • a duplex (108 and/or 110) is bound by the antibody or fragment 118, as shown at 114 and 112
  • the sensor cell can produce light which can be detected to indicate presence of the target RNA or DNA in the sample.
  • the sensor cell and the RNA-DNA duplex can be contacted in a liquid matrix.
  • liquid matrix can include, but is not limited to, homogeneous liquid including organic (alcohols, benzene, acids etc.) and inorganic (water etc.) liquid, and heterogeneous liquid constituting one or more organic or inorganic solutes or liquids, which is or are dissolved or mixed or suspended into another organic or inorganic liquid.
  • a solid matrix can be incorporated to facilitate higher assay sensitivity. Examples of solid matrices include, but are not limited to, plates, slides, dishes, beads, particles, cups, strands and chips.
  • the given liquid matrix can be used to transfer a duplex and biosensor cell and to provide a platform for signal generation.
  • a solid surface can be introduced to strategically concentrate duplex target in order to obtain higher sensitivity.
  • duplexes can be captured first immunologically or mechanically. The captured cells can then presented to the sensor cells for detection.
  • Biomarker cells include biomarker cells for use with the BioFlash® system based on the CANARY® (Cellular Analysis and Notification of Antigen Risks and Yields) technology (Innovative Biosensors, Inc., Rockville, MD).
  • BioFlash® can be used to rapidly detect the presence of a RNA-DNA duplex by detecting the binding of the RNA-DNA duplex with a sensor cell comprising a RNA-DNA duplex binding antibody or antibody fragment.
  • the BioFlash® system can comprise an engineered biosensor expressing membrane bound RNA-DNA duplex specific antibodies and a calcium sensitive bioluminescent molecule. Binding of the antibody by a RNA-DNA duplex leads to elevation of intracellular calcium and light emission.
  • the amplified light output can be detected by, for example, using a luminometer.
  • a RNA-DNA duplex to be detected binds to the antibody on the surface of a sensor cell, calcium ions move into the cytosol as described in Wilson ct al., J Exp Med 166:601-606 (1987).
  • the increased cytosolic calcium concentration causes an emitter molecule to emit a photon, which can be read by the optical detector.
  • a suitable emitter molecule is any molecule that emits a photon in response to elevated cytosolic calcium concentrations, including bioluminescent and fluorescent molecules.
  • bioluminescent aequorin protein is described in Button et al., Cell Calcium 14:663-671 (1993); Shimomura et al, Cell Calcium 14:373-378 (1993); and Shimomura, Nature 227: 1356-1357 ( 1970).
  • Aequorin generates photons by oxidizing coelenterazine, a small chemical molecule. Coelenterazine diffuses through cellular membranes, so coelenterazine or an analog thereof can be added to the culture medium surrounding the cells.
  • genes encoding enzymes that make coelenterazine can be introduced into the cells.
  • bio luminescent green fluorescent protein GFP
  • the cell cytosol contains both GFP and aequorin.
  • aequorin donates energy to GFP in an emissionless energy transfer process.
  • GFP then emits the photon.
  • the emitter molecule can be a calcium-sensitive fluorescent molecule (e.g., indo-1) which is illuminated by a wavelength of light suitable to induce fluorescence.
  • Aequorin, or any other emitter molecule can be introduced into the cell by methods well know in the art. If the emitter molecule is a protein (as is the case with aequorin), the cell can contain an expression vector encoding the protein (i.e., a nucleic acid or virus which will produce the emitter molecule when introduced into a cell). An expression vector can exist extrachromosomally or integrated into the cell genome.
  • a sensor cell with surface-bound antibodies can be cither prokaryotic or eukaryotic. Upon binding of a KNA-DNA duplex to the antibodies, the cell mobilizes calcium ions into the cytosol.
  • a sensor cell is a B cell (i.e., a B cell from a cold or warm-blooded vertebrate having a bony jaw) which can be genetically engineered to express one or more surface-bound monoclonal antibodies. It also can be produced by, for example, immunizing an animal with the antigen to be detected and harvesting the B cell from the immunized animal. The harvested B cells can be further immortalized and screened for production of a surface monoclonal antibody specific for the antigen to be detected. The B cells are optionally genetically engineered to express a calcium sensitive bioluminescent cytosolic molecule. Another useful cell type that can be used is a fibroblast that optionally can be adhered to a substrate or device.
  • fibroblasts do not contain the signal transduction machinery to transfer a signal from the cytoplasmic portion of a surface antibody via a signal cascade to calcium stores in the cell.
  • a chimeric surface antibody can be expressed in the fibroblast.
  • This chimeric antibody contains a cytoplasmic amino acid sequence derived from a polypeptide (e.g., a fibroblast growth factor receptor) that can transduce a signal from the inner surface of the plasma membrane of the fibroblast to intracellular calcium stores.
  • a polypeptide e.g., a fibroblast growth factor receptor
  • a similar strategy using chimeric antibodies can be employed for any other non B-cell type.
  • the fibroblast can also be genetically engineered to express a calcium sensitive bioluminescent cytosolic molecule.
  • Growth of the cell can be controlled by any means well known in the art, including providing anti -mitotic drugs (e.g., ⁇ -amanitin) or growth factors (e.g., fetal bovine serum) in the medium.
  • Cells can also be genetically engineered to grow at a determined rate.
  • any cell can be used where binding of a RNA- DNA duplex to the antibody on the surface of the cell leads to an increase in calcium concentration in the cytosol.
  • the cell can be a non-living, manufactured unit as long as it satisfies the above factors.
  • An antibody that specifically binds to a RNA-DNA duplex to be detected is a molecule that binds to the RNA-DNA duplex, but docs not substantially bind other antigens or epitopes in the sample.
  • the antibody does not substantially bind an RNA or DNA that is not duplexed.
  • Such antibodies can be chimeric (i.e., contain non-antibody amino acid sequences or contain portions from a different species) or single chain (i.e., the complementarity determining region of the antibody is formed by one continuous polypeptide sequence).
  • Polyclonal cells expressing antibodies can be prepared by immunizing a suitable animal with the antigen to be detected.
  • the cells producing antibody molecules directed against the antigen can be isolated from the animal (e.g., from the blood) and further purified by well-known techniques, such as panning against an antigen-coated petri dish.
  • Surface antibody-producing cells can be obtained from an animal and used to prepare a monoclonal population of cells producing surface antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al., Nature 256:495-497 (1975); Kozbor et al., Immunol Today 4:72 (1983); or Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., pp.
  • Any protocol used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a cell producing a surface monoclonal antibody (see, e.g., Current Protocols in Immunology, supra; Galfre et al., Nature 266:55052, 1977; Kenneth, In Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y., 1980; and Lerner, Yale J Biol Med 54:387-402 (1981).
  • a nucleic acid encoding a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the antigen to thereby isolate immunoglobulin library members that bind the antigen.
  • Kits for generating and screening phage display libraries arc commercially available.
  • examples of methods and reagents particularly amenable for use in generating and screening an antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.
  • the specific sequence can be cloned into any suitable nucleic acid expresser and transfected into a cell such as a fibroblast.
  • the expresser can also encode amino acids operably linked to the antibody sequence as appropriate for the cell which is to express the antibody.
  • the cytoplasmic transmembrane sequence of a fibroblast growth factor receptor can be linked to a single-chain antibody specific for the antigen to be detected, so that the cell mobilizes calcium when contacted with the antigen.
  • antibody fragment means a binding fragment of an antibody. Binding fragments include single chain fragments, Fv fragments and Fab fragments.
  • the term Fab is refers generically to double chain binding fragments of intact antibodies having at least substantially complete light and heavy chain variable domains sufficient for antigen-specific bindings, and parts of the light and heavy chain constant regions sufficient to maintain association of the light and heavy chains (e.g., including Fab' and F(ab') 2 fragments).
  • Fab fragments can be formed by complexing a full-length or substantially full-length light chain with a heavy chain comprising the variable domain and at least the CHl domain of the constant region. While various antibody fragments can be obtained by digesting an antibody, one of skill in the art will appreciate that such fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody fragment includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies ⁇ e.g., single chain Fv).
  • Single chain antibodies or fragments are also useful in the described methods. Methods for producing single chain antibodies are described, for example, in U.S. Pat. No.
  • Monoclonal antibodies can be prepared by any technique that provides for the production of antibody molecules by continuous cell lines in culture, including the hybridoma technique originally developed by Kohlcr and Milstein ⁇ Nature (1975) 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et a!., Immunology Today (1983) 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). Monoclonal antibodies also can be produced in germ-free animals as was described in U.S. Pat. No. 5,091,512. The antibodies and antibody fragments can be humanized or fully human ⁇ i.e., produced by transgenic animals that make human antibodies).
  • the antibodies and antibody fragments described herein can be screened for binding to a RNA-DNA duplex.
  • the terms bind, binds, and binding, when referring to an antibody or other binding moiety, are intended to indicate a binding reaction or affinity that is determinative of the presence of a target antigen (i.e., a RNA-DNA duplex), optionally, in the presence of a heterogeneous sample, population of proteins and/or other biologies.
  • a target antigen i.e., a RNA-DNA duplex
  • the specified binding moieties bind preferentially to a particular target antigen ⁇ i.e., a RNA-DNA duplex) and do not bind in a significant amount to other components present in a test sample.
  • a variety of immunoassay formats may be used to select antibodies or fragments that are specifically immunoreactive with a RNA-DNA duplex.
  • solid-phase ELTSA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an antigen.
  • a specific or selective reaction is at a statistically significant level above the background signal. For example, at least 1.5 to twice the background signal or noise or more than 10 times the background signal or noise.
  • Specific binding between an antibody or other binding agent and an antigen generally means a binding affinity of at least 10 6 M "1 .
  • Specific binding affinity include, but are not limited to at least 10 7 M "1 , at least 10 s M “1 , at least 10 9 NT 1 , and at least 10 10 M '1 .
  • Specific binding between an antibody or other binding agent and an antigen can also be described in terms of their dissociation constant KD.
  • the antibodies and antibody fragments described herein can bind with a KD of at least 1 ⁇ M, at least 500 ⁇ M, at least 300 ⁇ M, at least 100 ⁇ M, at least 50 ⁇ M, at least 30 ⁇ M, at least 10 ⁇ M, or at least 3 ⁇ M.
  • Methods for the detection of a RNA-DNA duplex in a sample include contacting the sample with an antibody or antibody fragment that binds to the RNA- DNA duplex.
  • Antibodies that bind a RNA-DNA duplex and methods of making the same are known in the art.
  • United States Patent 4,833,084 describes a monoclonal antibody, secreted by hybridoma ATCC HB 8730, specific for a RNA- DNA duplex.
  • Fliss et al., Production and Characterization of Anti-DNA- RNA Monoclonal Antibodies and Their Application in Listeria Detection, Appl and Envt Micro (1993) 59(8): 2698-2705 describes production of RNA-DNA duplex antibodies.
  • kits can comprise a container for containing the antibody or antibody fragment.
  • a kit can also contain components necessary to perform the assays described above or other assays.
  • the kit can comprise sensor cells such as those described above.
  • kits can further include apparatus for collecting, storing, and/or disposing of a sample.
  • Kits as described herein can include instructions, for example, for performing an assay or preparing a sample.
  • Such kits can contain one or more apparatus, matrix (liquid or solid) and chemical reagent needed to perform an assay or contain only certain elements of an assay.
  • RNA-DNA duplex For example, if a particular modification of a method of detecting a RNA-DNA duplex or is disclosed and discussed and a number of modifications that can be made to the method of detecting a RNA-DNA duplex are discussed, each and every combination and permutation of the RNA-DNA duplex and the detection method are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.
  • a hybridoma expressing antibody that specifically binds to a RNA-DNA duplex was obtained.
  • the antibody was expressed on the surface of the B-CeIl to form a CANARY® sensor cell.
  • Target RNA (or DNA) and complementary DNA (or RNA) were hybridized together.
  • a 2Kb length RNA-DNA duplex and a short 30 base pair length RNA-DNA oligo duplex were prepared.
  • the hybridized RNA-DNA duplexes were presented to the sensor cell to perform a CANARY® assay.
  • the CANARY® assay was carried out in a liquid matrix, which is the conventional matrix for a standard CANARY® assay.
  • Antibodies expressed on the B-cell sensor cell surface bound the RNA-DNA duplexes and triggered a constitutes of intracellular chemical and biochemical changes resulting in detectable photon emission. The photon emission was detected using standard optical equipment.
  • E-PAP polymerase
  • lO ⁇ l 25mM MnC12 lO ⁇ l ATP Solution
  • 4 ⁇ l E-PAP was added and mixed gently. The mixture was incubated 45 minutes at 37 0 C and transferred to ice. lO ⁇ l ammonium acetate was added and mixed. Extraction was performed with lOO ⁇ l phenol/chloroform/isoamyl alcohol by vortexing and spinning 5 minutes at 14K. Aqueous portion was transferred to a new tube and 1 volume isopropanol was added and mixed well. Chill -2O 0 C ON. The mixture was spun for 15 minutes at 14K.
  • Asymmetric PCR was performed by limiting the concentration of the kinased primer.
  • the strand primed by the kinased primer was preferentially degraded by lambda exonuclease 111 to obtain enriched ssDNA.
  • Kinased primer was prepared by incubating forward primer petpbp2a f xhol at lOOpmol with l ⁇ l Polynucleotide Kinase (PNK), l ⁇ l 1OX PNK Buffer, l ⁇ l 1OmM dATP, dH2O to lO ⁇ l for 30 minutes at 37 0 C. Enzyme was heat killed for 20 minutes at 65 0 C. Primer was diluted to lpmol/ ⁇ l for use.
  • Amplification was performed at Ix (92 0 C 2 minutes), 1Ox (92 0 C 10 seconds, 5O 0 C 15 seconds, 68 0 C 30 seconds), 2Ox (92 0 C 10 seconds, 5O 0 C 15 seconds, 68 0 C 30 seconds + 5 seconds per cycle), Ix (68 0 C 7 minutes) and 8 0 C hold.
  • PCR product was column purified using Qiaquick® PCR Purification Kit (Qiagen #28104) (Hilden, DE) using manufacturer's spin column instructions and elute in 35 ⁇ l dH2O. A check of 3.5 ⁇ l on 1.5% agarose/TAE gel was performed.
  • Lambda Exonuclease III Digestion (NEB #M0262S) 40 ⁇ l PCR product, lO ⁇ l 1OX Exo Reaction Buffer, 2 ⁇ l lambda exonuclease III, 48 ⁇ l dH2O were mixed and split into 8 PCR tubes ( ⁇ 12.5 ⁇ l per tube). The tubes were incubated 30 minutes at 37 0 C and 10 minutes at 75 0 C to heat kill enzyme. The samples were column purified using Qiaquick® PCR Purification Kit using manufacturer's spin column instructions and elute in 50 ⁇ l dH2O (Qiagen, Hilden, DE). 5 ⁇ l was checked on 1.5% agarose/lX TAE gel and concentration was determined by reading OD 260/280 nm in a spectrophotometer.
  • RNA:DNA--2 ⁇ l in 250 ⁇ l AB (f.c. lOclO RNA hybridized to 10c8 ssDNA or dilutions thereof)
  • RNA only-2 ⁇ l in 250 ⁇ l AB (f.c. lOel O RNA following above hybridization conditions)
  • ssDNA only--2 ⁇ l in 250 ⁇ l AB (f.c. 10e8 DNA following above hybridization conditions)
  • PIasmid (dsDNA) only-2 ⁇ l in 250 ⁇ l AB (f.c. 10e8 plasmid DNA following above hybridization conditions)
  • Biotinylated Oligo-Streptavidin beads complex was prepared as follows: Mix the biotinylated RNA oligos with DNA or the biotinylated DNA oligos with RNA in 0.1 or 1 X transcription buffer and water. Incubate for 5 minutes at 95 0 C followed by 30 minutes at 50 0 C. Add lO ⁇ g beads per sample and incubate 30 minutes at room temperature.
  • HCDNAOLIGO 5'-/5Biosg/CCG ACG CAG CAC TTG AGA CCA CAC CAT GCA ACT ACA TAC-(fam) HCRNA: 5'-rGrUrA rUrGrU rArGrU rUrGrC rArUrG rGrUrG rUrGrG rUrCrU rCrArA rGrUrG rCrUrG-3'
  • RNA/DNA target and select nucleic acid controls Two small scale B cell preparations (SS19 and SS21) of three clones (C21- 11 A, B and C) were made to evaluate the response to full length RNA/DNA target and select nucleic acid controls. Protocol: Samples were prepared by loading with coelenterazine according to standard CANARY® (Innovative Biosensors, Rockville, MD) small scale protocol.
  • Targets were full-length RNA alone or exo/ssDNA alone or full-length RNA/DNA hybridized in 0.1X transcription buffer then diluted in 250 ⁇ l assay buffer. The concentration of all conditions was RNA/DNA: 10el0/10e8 copies/assay. Samples were spun for 8 seconds. Integral RLU values are based on 40-50 seconds. A Signal to Noise (S :N) ratio of >3 was considered positive.
  • Protocol Samples were prepared following the above mentioned protocols. Targets were constituted using various combination of (+/- biotin)RNA oligo, (+/- biotin)DNA oligo + beads or in combination + beads in 250 ⁇ l assay buffer. Also FL- RNA+DNA was used as control. The concentration of all conditions was RNA/DNA: 10el0/10e8 copies/assay. Samples were normalized to 1OK cells and were spun for 8 minutes prior to assaying.
  • Results indicate good signal with biotinylated DNA/RNA oligo+beads labeled as bDR (biotinylated DNA+ RNA) or bRD (biotinylated RNA+ DNA).
  • bDR biotinylated DNA+ RNA
  • bRD biotinylated RNA+ DNA
  • the previously described 2 kb long RNA-DNA duplex was used as positive control (AB+RD) and yielded robust signal. No signal was seen with mock beads (AB+beads) and with RNA oligo alone or DNA oligo alone controls.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne des procédés et des compositions de détection d’un duplex ARN-ADN dans un échantillon.
PCT/US2009/051657 2008-07-24 2009-07-24 Détection de molécules duplex arn-adn Ceased WO2010011901A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US8336008P 2008-07-24 2008-07-24
US61/083,360 2008-07-24

Publications (1)

Publication Number Publication Date
WO2010011901A1 true WO2010011901A1 (fr) 2010-01-28

Family

ID=41570617

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/051657 Ceased WO2010011901A1 (fr) 2008-07-24 2009-07-24 Détection de molécules duplex arn-adn

Country Status (1)

Country Link
WO (1) WO2010011901A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4732847A (en) * 1981-06-09 1988-03-22 University Of Hawaii Monoclonal antibodies for DNA-RNA hybrid complexes and their uses
US6248542B1 (en) * 1997-12-09 2001-06-19 Massachusetts Institute Of Technology Optoelectronic sensor
US20040180362A1 (en) * 1998-02-06 2004-09-16 Lazar James G. Immunological detection of RNA:DNA hybrids on microarrays
US20080131883A1 (en) * 2004-11-03 2008-06-05 Iris Molecular Diagnostics, Inc. Homogeneous Analyte Detection

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4732847A (en) * 1981-06-09 1988-03-22 University Of Hawaii Monoclonal antibodies for DNA-RNA hybrid complexes and their uses
US6248542B1 (en) * 1997-12-09 2001-06-19 Massachusetts Institute Of Technology Optoelectronic sensor
US20040180362A1 (en) * 1998-02-06 2004-09-16 Lazar James G. Immunological detection of RNA:DNA hybrids on microarrays
US20080131883A1 (en) * 2004-11-03 2008-06-05 Iris Molecular Diagnostics, Inc. Homogeneous Analyte Detection

Similar Documents

Publication Publication Date Title
Zheng et al. Simultaneous and ultrasensitive detection of foodborne bacteria by gold nanoparticles-amplified microcantilever array biosensor
US9157116B2 (en) Combinatorial amplification and detection of nucleic acids
KR102458825B1 (ko) 핵산 검출 방법
Frank et al. Application of enzyme bioluminescence for medical diagnostics
JPH10508741A (ja) 核酸−プローブ標的ハイブリッド検出の感度上昇のための増幅方法
AU2009317878A1 (en) Analyte detection assay
Gaa et al. Versatile and rapid microfluidics-assisted antibody discovery
Liu et al. CRISPR-Cas12a coupled with universal gold nanoparticle strand-displacement probe for rapid and sensitive visual SARS-CoV-2 detection
EP2865764B1 (fr) Procédé de détection d'un acide nucléique et utilisation d'un kit de détection afférent
EP1766049B1 (fr) Procede de detection d'analytes dans un echantillon
JP3130538B2 (ja) 高感度蛍光アッセイ
CN107828861A (zh) 基于微流控芯片和g‑四链体‑血红素dna酶检测循环核酸用试剂盒及其制备方法和应用
JPWO1997047968A1 (ja) 高感度蛍光イムノアッセイ
CN105385753A (zh) 基于核酸适配体检测水胺硫磷的电化学传感器及其制备方法
CN102230017B (zh) 人类血小板抗原基因分型液相芯片及其检测方法
JP2009296948A (ja) Ｐｃｒ用プライマー、標的核酸の検出方法及び標的生体分子の検出方法
AU773046B2 (en) Test system for detecting different markers, and production and use thereof
WO2010011901A1 (fr) Détection de molécules duplex arn-adn
JP7021949B2 (ja) 抗体検出法及び抗体検出系
AU2007297164A1 (en) Nucleic acid detection using flow through methods
WO2006129770A1 (fr) Procede de detection d’acide nucleique
CN107976548B (zh) 基于微流控芯片和g-四链体-血红素dna酶的检测凝血酶用试剂盒及其制备方法和应用
CN110904249A (zh) 一种细菌耐药基因量子点芯片核酸检测试剂盒及检测方法
EP2105741B1 (fr) Utilisation d'un virus exprimant un groupe caractéristique de liaison pour mesurer des analytes dans un échantillon
Cansız et al. A sandwich-type DNA array platform for detection of GM targets in multiplex assay

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09801051

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09801051

Country of ref document: EP

Kind code of ref document: A1