WO1991002088A1 - Compositions renfermant des lactames et procedes utiles pour l'hybridation d'acides nucleiques - Google Patents

Compositions renfermant des lactames et procedes utiles pour l'hybridation d'acides nucleiques Download PDF

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WO1991002088A1
WO1991002088A1 PCT/US1990/004152 US9004152W WO9102088A1 WO 1991002088 A1 WO1991002088 A1 WO 1991002088A1 US 9004152 W US9004152 W US 9004152W WO 9102088 A1 WO9102088 A1 WO 9102088A1
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nucleic acid
hybridization
pyrrolidone
carbons
lactam
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PCT/US1990/004152
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English (en)
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Jeffrey Van Ness
Nicolaas M. J. Vermeulen
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Microprobe Corporation
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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
    • C12Q1/6832—Enhancement of hybridisation reaction
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • This invention relates to compositions and assay methods for the hybridization and extraction of nucleic acids.
  • this invention relates to compositions and methods to release nucleic acids from cells in complex biological samples or specimens while simultaneously hybridizing complementary nucleic acids released during lysis.
  • lactams preferably pyrrolidones
  • lactams which promote nucleic acid base pairing
  • highly sensitive assays for ribonucleic acid are also described that employ a heating step.
  • Organic solvents such as phenol and chloroform are traditionally used in techniques employed to isolate nucleic acid from procaryotic and eucaryotic cells or from complex biological samples. Nucleic acid isolations typically begin with an enzymatic digest performed with proteases followed by cell lysis using ionic detergents and then extraction with phenol or a phenol/chloroform combination. The organic and aqueous phases are separated and nucleic acid which has partitioned into the aqueous phase is recovered by precipitation with alcohol.
  • phenol or a phenol/chloroform mixture is corrosive to human skin and is considered as hazardous waste which must be carefully handled and properly discarded. Further, the extraction method is time consuming and laborious. Marmur, J. Mol. Biol.
  • chaotropic agents such as guanidine thiocyanate (GnSCN) are widely used to lyse and release nucleic acid from cells into solution, largely due to the fact that the chaotropic salts inhibit nucleases and proteases.
  • GnSCN guanidine thiocyanate
  • Nucleic acid hybridization is a known and documented method for identifying nucleic acids. Hybridization is based on base pairing of complementary nucleic acid strands. When single stranded nucleic acids are incubated in appropriate buffer solutions, complementary base sequences pair to form double stranded stable molecules. The presence or absence of such pairing may be detected by several different methods well known in the art. Most hybridization assays previously described involve multiple steps such as the hybridization technique described by Dunn & Hassell in Cell, Vol. 12, pages 23-36 (1977) . Their assay is of the sandwich-type whereby a first hybridization occurs between a "target" nucleic acid and a "capture” nucleic acid probe which has been immobilized on a solid support.
  • a second hybridization then follows where a "signal" nucleic acid probe, typically labelled with a radioactive isotope, hybridizes to a different region of the immobilized target nucleic acid.
  • the hybridization of the signal probe may then be detected by, for example, autoradiography.
  • Ranki et al. in U.S. Patent No. 4,486,539 and 4,563,419 describe sandwich-type assays which first require steps to render nucleic acids single stranded and then the single stranded nucleic acids are allowed to hybridize with a nucleic acid affixed to a solid carrier and with a nucleic acid labelled with a radioisotope.
  • the Ranki et al. assay requires the nucleic acid to be identified or targeted in the assay to be first rendered single stranded.
  • Hybridization media and extraction solutions which avoid the problems encountered with the use of chaotropic agents and toxic organic solvents, but which also yield sensitive assays would be desirable. Further, procedures which minimize the handling of reagents and samples would be advantageous.
  • This invention relates to novel methods for the release of nucleic acids from cells in complex biological samples or specimens to prepare and make available the nucleic acid material present for a hybridization assay or for extraction. Novel methods for hybridization of nucleic acids are also presented. In particular, methods are described for the hybridization of nucleic acid from a sample suspected of containing a target nucleic acid of interest wherein the sample is combined with a hybridization medium comprising a lactam which promotes and enables nucleic acid pairing when a nucleic acid complementary to the target nucleic acid is introduced. The extent of hybridization of the complementary nucleic acid to the target nucleic acid is then determined.
  • the concentration of the lactam is preferably about 5 to about 70% of the hybridization medium and is most preferably 2-pyrrolidone, N-ethy1-2-pyrrolidone, N-cyclohexyl-2- pyrrolidone, N-dodecyl-2-pyrrolidone, N-methyl-2-pyrrolidone, N-hydroxyethyl -2-pyrrolidone, N-methyl-2-piperidone, 2-e- caprolactam, N-methyl-2-caprolactam, 2-piperidone (or 2-5- valerolactam) or N-(4-hydroxybenzyl)pyrrolidone.
  • hybridization may be done completely at room temperature and in one easy step with all reagents precombined. This is particularly true if open regions of ribosomal RNA (rRNA) are used as the target nucleic acids.
  • rRNA ribosomal RNA
  • lactams used in the present invention are of a very low order toxicity and are not corrosive to human tissue.
  • __ when used in extraction procedures preceding hybridization they are useful in removing background interference. They retain most of the solvent properties of phenol or phenol/chloroform and are uniquely suited to the application of nucleic acid isolation and/or fractionation of biological macromolecular complexes. When used in extraction procedures, they are substantially less expensive than the commonly employed organic solvents. Further, the use of lactams avoids problems relating to the incompatibility of chaotropes with ionic detergents during isolation of nucleic acids from chaotropic salt solutions.
  • This invention relates to novel means and methods for releasing nucleic acids from a complex biological mixture containing nucleic acids and non-nucleic acids and promoting nucleic acid pairing. Highly sensitive hybridization assays are also described.
  • the methods of the present invention enable one to easily process a biological sample containing nucleic acids by promoting the lysis of cells in the sample and promoting the hybridization of nucleic acids suspected of being present in the cells.
  • the methods of the present invention further enable one to readily assay for a nucleic acid suspected of being present in cells, i.e., a target nucleic acid.
  • Such methods include lysing the cells in a hybridization medium comprising a lactam, contacting the lysate under hybridization conditions with a nucleic acid having a nucleotide sequence substantially complementary to a sequence suspected to be present in the cells, and determining the extent of hybridization.
  • target nucleic acid means the nucleotide sequence of deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) or riboso al ribonucleic acid (rRNA) whose presence is of interest and whose presence or absence is to be detected for in the hybridization assay.
  • the nucleic acid sample of interest will be one which is suspected of containing a particular target nucleic acid, such as one known to be associated with a particular microorganism, whose presence or absence is of interest.
  • the target nucleic acid may be provided in a complex biological mixture of nucleic acid (RNA, DNA and/or rRNA) and non-nucleic acid.
  • target nucleic acids of primary preference are RNA molecules and, in particular, open regions of rRNA which have minimal secondary or tertiary interactions with adjacent nucleotides, such as on the 16s or 23s rRNA as described in commonly assigned U.S. Patent Application No. 142,106, which is incorporated by reference herein.
  • target nucleic acids of choice are double stranded or otherwise have significant secondary and tertiary structure, they may need to be heated prior to hybridization. In this case, heating may occur prior to or after the introduction of the nucleic acids into the hybridization medium containing the lactam. It may also be desirable in some cases to extract the nucleic acids from the complex biological samples prior to the hybridization assay to reduce background interference by any methods known in the art. Particularly preferable are those extraction procedures utilizing lactams which are disclosed in commonly assigned U.S. Patent Application No. 07/384,367 filed July 24, 1989, which is incorporated by reference herein.
  • the hybridization and extraction methods of the present invention may be applied to a complex biological mixture of nucleic acid (RNA and/or DNA) and non-nucleic acid.
  • a complex biological mixture includes a wide range of eucaryotic and procaryotic cells, including protoplasts; or other biological materials which may harbor polynucleotide target nucleic acids.
  • the methods are thus applicable to tissue culture animal cells, animal tissue (e.g., heart, liver or brain, homogenized in lysis buffer) , blood cells, reticulocytes, lymphocytes, plant cells or other cells sensitive to osmotic shock and cells of bacteria, yeasts, viruses, mycoplasmas, protozoa, rickettsia, fungi and other small microbial cells and the like.
  • the assay and isolation procedures of the present invention are useful, for instance, for detecting non-pathogenic or pathogenic microorganisms of interest. By detecting specific hybridization between nucleotide probes of a known source and nucleic acids resident in the biological sample, the presence of the microorganisms may be established.
  • Lactams are a class of a group of organic cyclic compounds containing the - NH-CO- group in the ring. Such compounds are typically formed by the elimination of water from the amino and carboxyl groups of a noncyclic compound.
  • the particular substituents on the lactam ring are not critical to this invention. It is, however, important that the lactam maintain hydrogen bonding capacity and water solubility. Thus, substituents of more than eight carbons, for example, would be less preferred than substituents with less than eight carbons which would be more water soluble. This is especially true if the preferred substitutents contain additional polar groups.
  • the lactam will be of the general formula including racemic mixtures and optically active isomers:
  • R is selected from the group consisting of: hydrogen, alkyl of 1 to 20 carbons, aryl of 6 to 10 carbons, arylalkyl of 7 to 20 carbons, hydroxyarylalkyl of 7 to 20 carbons, alkylcarboxyamide of 1 to 20 carbons, N-mono-substituted alkylcarboxyamide, N, N-dialkyl-substituted alkylcarboxyamide, alkylcarboxylate of 2 to 20 carbons, acyl of 2 to 20 carbons, cycloalkane of 4 to 20 carbons, hydroxyalkyl, and cyanoalkyl; and where the R substituents can be the same or different and selected from a group consisting of hydrogen, halogen and alkyl of 1 to 5 carbons such that the total carbon number for all R substituents does not exceed 20; with the further provision that the total carbon number of the R
  • Pyrrolidones, piperidones and caprolactams are lactams which are particularly preferred.
  • lactams examples include 2-pyrrolidone, N-methyl-2-pyrrolidone, N-hydroxyethyl-2-pyrrolidone, N-cyclohexyl-2-pyrrolidone, N-dodecyl-2-pyrrolidone, N-ethyl- 2-pyrrolidone, N-methyl-2-piperidone, 2-e-caprolactam, N-methyl-2-caprolactam, 2-piperidone (or 2- ⁇ S-valerolactam) and N-(4-hydroxybenzyl)pyrrolidone.
  • These are commercially available from ⁇ the GAF Chemicals Corporation (a subsidiary of GAF Corporation, Wayne, New Jersey) and/or Aldrich Chemical Company (Milwaukee, Wisconsin) .
  • Alkyl refers to an aliphatic hydrocarbon radical
  • -(CH 2 ) n CH 3 either branched or unbranched such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, dodecyl or the like.
  • Aryl refers to a radical derived from an aromatic hydrocarbon by removal of one hydrogen atom such as phenyl, ⁇ -napthyl, ⁇ -napthyl, biphenyl, anthryl and the like.
  • Arylalkyl refers to an alkyl radical as defined above joined to an aryl radical.
  • Alkylcarboxyamide refers to a radical, -(CH 2 ) n -CONH 2 .
  • Hydroxyarylalkyl refers to an arylalkyl radical where the aryl radical is an hydroxyaryl.
  • N,N-dialkyl-substituted alkylcarboxyamide refers to a radical
  • Acyl includes any organic radical derived from an organic acid, such as a carboxylic acid by elimination of the hydroxyl group. It is represented by the formula R 6 -C0, wherein R 6 can be as defined in U.S. Patent No. 4,665,067 at column 2, line 31 through column 12, line 32, which is incorporated by reference herein. It is preferred that R 6 be an alkyl of 1 to 20 carbons or a cycloalkyl.
  • Cycloalkane or cycloalkyl refers to a radical of a saturated hydrocarbon in a ring structure such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyeloheptyl, adamantyl and the like. Cyanoalkyl refers to a radical of a cyano group, having the formula -C ⁇ N joined to an alkyl group, as defined above.
  • Halogen refers to chlorine, bromine, iodine or fluorine.
  • Alkali metal derivatives of pyrrolidone are readily formed by reaction with metal or from concentrated caustic soda and 2-pyrrolidone in acetone solution as described in "Acetylene Chemistry," J.W. Reppe P.B. Report, 18-852-s, CA. Meyer & Co., Inc., New York, NY (1949), 210 pp., incorporated by reference herein. Such procedure permits reaction with substituted alkyl, aryl or arylalkyl halides without opening the lactam ring, to give N-alkyl, N-aryl or N-arylalkyl pyrrolidones. See Method A on Figure IA.
  • N- oct 1-2-pyrrolidone and N-dodecy1-2-pyrrolidone are synthesized in this manner and belong to the N-alkyl-2-pyrrolidone family.
  • N-methyl-2-pyrrolidone, the lactam of 4-methylaminobutyric acid, is synthesized using the Reppe chemistry described above. Acylated lactams can also be chlorinated and brominated at elevated temperatures under the influence of light.
  • Lactams can react with the following reagents: a) Acid anhydrides or chlorides result in formation of N-acyl lactams; for example acetic anhydride forms from N- acety1-2-pyrrolidone. See Method B on Fig. IA. b) N-methylol-2-pyrrolidone results from reaction of formaldehyde and 2-pyrrolidone. c) N-(2-hydroxymethyl)-2-pyrrolidone is formed by reaction with ethylene oxide. See Method C on Fig. IA.
  • Method D on Fig. IA shows the reaction of butyrolactone in the presence of a moderate excess of ammonia under heat to yield 2-pyrrolidone.
  • Method F on Fig. IB shows the" same reaction using butyrolactone and alkylamine as the starting materials to yield an alkyl substituted lactam.
  • Method E on Fig. IA shows that lactams are easily produced from the pyrolysis of amino acids.
  • the reaction of hydrazoic acids with cyclic ketones yield the corresponding lactam as shown in method G on Fig. IB.
  • the commercially available technical grades of all pyrrolidones should be vacuum distilled prior to use with biological samples.
  • the lactams should be provided in concentrations in excess of 5% of the total volume of the hybridization medium or extraction solution up to a concentration of about 70%, preferably a concentration of 20% to 50%, most preferably a concentration of about 30% to 50%.
  • the lactams may be used singly or in combination.
  • Solutions containing a lactam or mixtures of the lactams are capable of effectively lysing procaryotic and eucaryotic cells while simultaneously promoting specific hybridization of nucleic acid probes to released endogenous nucleic acid.
  • the solutions need not contain any other component other than common buffers and detergents to promote lysing and solubilization of cells and nucleic acid hybridization.
  • organic solvents such as phenol and chloroform may be used in techniques employed to isolate nucleic acid.
  • organic solvents such as phenol or a phenol- chloroform combination are used to extract nucleic acid, using a phase separation. These methods may be used effectively with the lysis solutions of the present invention; however, an advantage of the methods of the present invention is that such toxic and tedious extraction methods are not necessary.
  • the combined hybridization media or extraction solution with the complex biological mixture itself may become biphasic with the non-nucleic acid material present in the organic phase and the nucleic acid solution present in the aqueous phase.
  • the combined solution is typically mixed and subjected to centrifugation and the nucleic acid is precipitated with ethanol.
  • the hybridization medium will contain standard buffers and detergents to promote lysing of cells.
  • a buffer such as sodium citrate, Tris HC1, PIPES or HEPES, preferably Tris-HCl at a concentration of about 0.05 to 0.1M can be used.
  • the hybridization medium will preferably also contain about 0.05 to 0.5% of an ionic or nonionic detergent, such as sodium dodecylsulfate (SDS) or Sarkosyl (Sigma Chemical Co. , St. Louis, Missouri) and between 1 to lOmM EDTA.
  • SDS sodium dodecylsulfate
  • Sarkosyl Sigma Chemical Co. , St. Louis, Missouri
  • volume exclusion agents which include a variety of polar water-soluble or swellable agents, such as anionic polyacrylate or polymethacrylate, and charged saccharidic polymers, such as dextran sulfate and the like.
  • Specificity or the stringency of hybridization may be controlled, for instance, by varying the NaCl concentration which is typically between 0 to IM NaCl.
  • Chaotropic agents which disturb the secondary and tertiary structure of proteins for example, guanidine salts such as guanidine hydrochloride (GnHCl) and thiocyanate (GnSCN) , or urea, lithium chloride and other thiocyanates may be used in combination with the lactams to dissociate nucleic acids and inhibit nucleases.
  • GnHCl guanidine hydrochloride
  • GnSCN thiocyanate
  • urea lithium chloride and other thiocyanates
  • a nucleic acid substantially complementary to the target nucleic acid will be introduced in the hybridization process.
  • a nucleic acid substantially complementary to the target nucleic acid is a polynucleotide or oligonucleotide containing naturally occurring nucleotides or their analogs, such as 7-deazaguanosine or inosine, sufficiently complementary to hybridize with the target nucleic acid such that stable and specific binding occurs between the target and the complementary nucleic acid. Therefore, the complementary nucleic acid sequence need not reflect the exact sequence of the target nucleic acid.
  • a non- complementary nucleotide fragment may be attached to a complementary nucleotide fragment or alternatively, non- complementary bases or longer sequences can be interspersed into the complementary nucleic acid, provided that the complementary nucleic acid sequence has sufficient complementarity with the sequence of the target nucleic acid to hybridize therewith forming a hybridization complex and further is capable of immobilizing the target nucleic acid to a solid support.
  • the degree of homology required for formation of a stable hybridization complex (duplex) varies with the stringency of the hybridization medium and/or wash medium. Oligonucleotide probes to rRNA are most preferred such as those described in commonly assigned U.S. Patent Application Serial No. 142,106, which is incorporated by reference herein.
  • the complementary nucleic acid may be present in a pre-prepared hybridization media or introduced at some later point prior to hybridization.
  • the hybridization medium is combined with the biological sample to facilitate lysis of the cells and nucleic acid pairing.
  • the volume of biological sample to the volume of the hybridization medium will be about 1:10.
  • hybridization methods of the present invention are that they be carried out on complex biological samples. However, minor mechanical or other treatments may be considered under certain circumstances. For example, it may be desirable to clarify the lysate before hybridization such as by slow speed centrifugation or filtration or to extract the nucleic acids first as described above.
  • the hybridization assay of the present invention can be performed by any method known to those skilled in the art or analogous to im unoassay methodology given the guidelines presented herein. Preferred methods of assay are the sandwich assays and variations thereof and the competition or displacement assay. Hybridization techniques are generally described in "Nucleic Acid Hybridization, A Practical Approach," Ed. Hames, B.D.
  • the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected.
  • a nucleic acid amplification system which multiplies the target nucleic acid being detected.
  • examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA , Cangene, Mississauga, Ontario) and Q Beta Replica ⁇ e systems.
  • PCR is a template dependent DNA polymerase primer extension method of replicating select sequences of DNA. The method relies upon the use of an excess of specific primers to initiate DNA polymerase replication of specific subsequences of a DNA polynucleotide followed by repeated denaturation and polymerase extension steps.
  • PCR system is well known in the art (see U.S. Patent Nos. 4,683,195 and 4,683,202).
  • PCR Protocols A Guide to Methods and Applications, ed. Innis, Gelfand, Sninsky and White, Academic Press, Inc. (1990) .
  • Reagents and hardware for conducting PCR are available commercially through Perkin-Elmer/Cetus Instruments of Norwalk, Connecticut.
  • LCR like PCR, uses multiple cycles of alternating temperature to amplify the numbers of a targeted sequence of DNA. LCR, however, does not use individual nucleotides for template extension.
  • LCR relies instead upon an excess of oligonucleotides which are complementary to both strands of the target region.
  • the LCR procedure begins with the ligation of two oligonucleotide primers complementary to adjacent regions on one of the target strands. Oligonucleotides complementary to either strand can be joined. After ligation and a second denaturation step, the original template strands and the two newly joined products serve as templates for additional ligation to provide an exponential amplification of the targeted sequences. This method has been detailed in Genomics, 4:560-569 (1989), which is incorporated herein by reference. As other amplification systems are developed, they may also find use in this invention.
  • the hybridization media and processes of the present invention are uniquely suited to a one-step assay.
  • the media may be pre-prepared, either commercially or in the laboratory to contain all the necessary components for hybridization.
  • the media could comprise a lactam, desired buffers and detergents, a capture nucleic acid bound to a solid support such as a microbead, and a signal nucleic acid.
  • This media then need only be combined with the sample containing the target nucleic acid at the time the assay is to be performed. Once hybridization occurs the hybridization complex attached to the solid support may be washed and the extent of hybridization determined.
  • Sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and labelled “signal" nucleic acid in solution. The clinical sample will provide the target nucleic acid. The "capture” nucleic acid and “signal” nucleic acid probe hybridize with the target nucleic acid to form a "sandwich” hybridization complex. To be effective, the signal nucleic acid cannot hybridize with the capture nucleic acid.
  • any solid surface can be used as a support for hybridization assays, including metals and plastics.
  • Two types of solid surfaces are generally available namely: a) Membranes, polystyrene beads, nylon, teflon, polystyrene/latex beads, latex beads or any solid support possessing an activated carboxylate, sulfonate, phosphate or similar activatable group are suitable for use as solid surface substratum to which nucleic acids or oligonucleotides can be immobilized.
  • Porous membranes possessing pre-activated surfaces which may be obtained commercially (e.g., Pall Immunodyne Immunoaffinity Membrane, Pall BioSupport Division, East Hills, NY, or I mobilon Affinity membranes from Millipore, Bedford, Mass.) and which may be used to immobilize capture oligonucleotides.
  • Microbeads, including magnetic beads, of polystyrene, teflon, nylon, silica or latex may be used.
  • Capture or signal nucleic acids for use in hybridization assays can be obtained from the entire sequence or portions thereof of an organism's genome, from messenger RNA, or from cDNA obtained by reverse transcription of messenger RNA. After isolation of genomic DNA or cDNA fragments, the fragments are typically inserted into a replication vector, such as lambda phage, pBR322, M13, or vectors containing the SP6 or T7 promoter and cloned as a li ⁇ brary in a bacterial host. Following appropriate screening procedures, a recombinant vector with the desired probe insert is isolated and labelled as described below. The vector is then grown in a suitable host. The probe and its vector are purified from the host cells by cell lysis and nucleic acid extraction. Following isolation, the probe can be purified away from the vector by digestion with selected restriction en- zymes and sequenced. Further isolation of the probe can be achieved fey using gel electrophoresis or high pressure liquid chromatography.
  • DNA probes are preferably chemically synthesized using commercially available methods and equipment.
  • the solid phase phosphoramidite method can be used to produce short probes of between 15 and 50 bases and have a molecular weight of less than 16,000 daltons. (Caruthers et al., Cold Spring Harbour Symp. Quant. Biol., 4_7:411-418 (1982), and Adams et al., J. Am. Chem. Soc.. 105:661 (1983).
  • nucleotide sequence When synthesizing a probe for a specific target, the choice of nucleotide sequence will determine the specificity of the test. For example, by comparing DNA sequences from several virus isolates, one can select a sequence for virus detection that is either type specific or genus specific. Comparisons of DNA regions and sequences can be achieved using commercially available computer programs.
  • the determination of the extent of hybridization may be done by any of the methods well-known in the art. If there is no detectable hybridization, the extent of hybridization is thus 0.
  • labelled signal nucleic acids are used to detect hybridization.
  • Complementary nucleic acids or signal nucleic acids may be labelled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 14 C, or 32 P-labelled probes or the like. The choice of radioactive isotope depends on research preferences due to ease of synthesis, varying stability, and half lives of the selected isotopes.
  • Other labels include ligands which bind to labelled antibodies, fluorophores,. chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labelled ligand. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.
  • Radioactive probes are typically made using commercially available nucleotides containing the desired radioactive isotope.
  • the radioactive nucleotides can be incorporated into probes by several means such as by nick translation of double-stranded probes; by copying single- stranded M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase in the presence of radioactive dNTP; by transcribing cDNA from RNA templates using reverse trans- criptase in the presence of radioactive dNTP; by transcribing RNA from vectors containing SP6 promoters or T7 promoters using SP6 or T7 RNA polymerase in the presence of radioactive rNTP; by tailing the 3' ends of probes with radioactive nucleotides using terminal transferase; or by phosphorylation of the 5' ends of probes using [ 32 P]-ATP and polynucleotide kinase
  • Non-radioactive probes are often labelled by indirect means.
  • a ligand molecule is covalently bound to the probe.
  • the ligand then binds to an anti-ligand moiecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • Ligands and anti-ligands may be varied widely. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labelled, naturally occurring anti- ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
  • Probes can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, AMPPD([3-(2'- spiroamantane)-4-methoxy-4-(3 'phosphoryloxy)-phenyl-1,2- dioxetane]) and 2,3-dihydrophthalazinediones, e.g., luminol.
  • the amount of labelled probe which is present in the hybridization medium or extraction solution may vary widely. Generally, substantial excesses of probe over the stoichiomgtric amount of the target nucleic acid will be employed " to enhance the rate of binding of the probe to the target DNA. Treatment with ultrasound by immersion of the reaction vessel into commercially available sonication baths can often times accelerate the hybridization rates.
  • the glass, plastic, or filter support to which the capture nucleic acid-target nucleic acid hybridization complex is attached is introduced into a wash solution typically containing similar reagents (e.g., sodium chloride, buffers, organic solvents and detergent) , as provided in the hybridization solution.
  • reagents e.g., sodium chloride, buffers, organic solvents and detergent
  • the time period for which the support is maintained in the wash solutions may vary from minutes to several hours or more.
  • Either the hybridization or the wash medium can be stringent. After appropriate stringent washing, the correct hybridization complex may now be detected in accordance with the nature of the label.
  • the probe may be conjugated directly with the label.
  • the probe with associated hybridization complex substrate is exposed to X-ray film.
  • the label is fluorescent
  • the sample is detected by first irradiating it with light of a particular wavelength. The sample absorbs this light and then emits light of a different wavelength which is picked up by a detector fPhysical Biochemistry, Freifelder, D., W.H. Freeman _ Co. (1982), pp. 537-542) .
  • the label is an enzyme
  • the sample is detected by incubation on an appropriate substrate for the enzyme.
  • the signal generated may be a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemi-luminescence.
  • the preferred label for probe assays generates a colored precipitate to indicate a positive reading.
  • alkaline phosphatase will dephosphorylate indoxyl phosphate which then will participate in a reduction reaction to convert tetrazolium salts to highly colored and insoluble formazans.
  • Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.
  • the binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • the label may also allow indirect detection of the hybridization complex.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label.
  • a chaotropic agent such as a salt of isothiocyanate (e.g., guanidine thiocyanate) does not provide for the complete disruption of protein and nucleic acid interactions, thus, preventing optimal hybridization.
  • a significant and surprising increase in hybridization does occur when heat is applied to the hybridization solution containing the chaotropic agent and target nucleic acid.
  • researchers have attempted to keep hybridization temperatures low to maintain stability of the reactants. See Cox et al., EP Application No. 84302865.5. Thus, it was surprising that heat would improve the sensitivity of the hybridization assay and do so so dramatically.
  • This invention also provides efficient methods for increasing the sensitivity of ribonucleic acid hybridization assays and for simplifying the steps of the assays.
  • the processes for conducting nucleic acid hybridizations wherein the target nucleic acid is RNA comprise heating a nucleic acid solution or sample to a temperature of about 60-100°C, preferably greater than 65 ⁇ C and most preferably to about 65°- 70 P C.
  • the nucleic acid solution will comprise a chaotropic agent, a target nucleic acid, and a nucleic acid substantially complementary to the target nucleic acid of interest.
  • the nucleic acid solution will be heated to fully disrupt the protein and nucleic acid interactions to maximize target nucleic acid hybridization.
  • the heating time is preferably about 1 to 10 minutes, most preferably about 5 minutes for solutions containing about 0.001 to 1000 ⁇ g of nucleic acid per ml.
  • the addition of the heating step increases the sensitivity of the hybridization assays regardless of whether lactams are included.
  • the solution is then cooled until the complementary nucleic acid has hybridized with the target nucleic acid to form a hybridization complex.
  • the cooling process and rate of cooling are not critical. Cooling may be achieved by simply allowing the solution to cool to room temperature.
  • a prepared reagent solution may be provided, for example, which would contain a chaotropic agent, other appropriate components such as buffers or detergents, a capture nucleic acid bound to a solid support, and a signal nucleic acid, both capable of hybridizing with a target nucleic acid.
  • a complex biological sample suspected of harboring a target nucleic acid can be directly combined with the pre-prepared reagent for hybridization, thus, allowing the hybridization to occur in .one step.
  • the combined solution is heated as described herein and then cooled until hybridization has occurred.
  • the resulting hybridization complex is then simply washed to remove unhybridized material and the extent of hybridization is determined.
  • Kits for the extraction of and hybridization of nucleic acids are also contemplated. Such kits would contain at least one vial containing an extraction solution or a hybridization medium which comprises a lactam at a concentration in excess of 5%. Detergents, buffer solutions and additional vials which contain components to detect target nucleic acids may also be included.
  • EXAMPLE I Lysis of bacterial and human cells with pyrrolidone-based solutions.
  • a lysis solution composed of 20% N-cyclohexyl-2- pyrrolidone, 20% N-hydroxymethyl-2-pyrrolidone, 10% N-dodecyl- 2-pyrrolidone, IM Guanidine-HCl, 50mM Tris, pH 7.6, 25mM EDTA, and 2% SDS was used to lyse 1 x 10 9 cells of Aa, Bi, Ec, Wr, Fn, Bg, and 1 x 10 6 cells of HeLa in 100 microliter volumes.
  • the solution was noted to clear within 10 seconds and phase contrast microscopy indicated the solubilization of all the cell types tested. No intact cell shapes were observed by light microscopy.
  • a solution composed of 20% N-cyclohexyl-2- pyrrolidone, 20% N-hydroxymethyl 2-pyrrolidone, 50mM Tris pH 7.6, 25mM EDTA and 1% SDS was used to test the ability of 32 P- labelled specific oligonucleotide probes (synthetic oligonucleotide probes in which the sequence is complementary to unique hypervariable regions of the 16s rRNA of the respective bacterium) to hybridize specifically to total nucleic acid from Aa, Bg, Bi, Ec, Fn, Wr and E. coli immobilized on Nytran filters (Schleicher and Schuell, Keene, New Hampshire) .
  • nytran slot Approximately 50 nanograms of total nucleic acid was immobilized on each nytran slot. 6.5 x 10 6 cpm of 32 P- labelled probe (approximately 65 nanograms) of Aa complementary probe (Aa4B) was added to 1ml of the solution described above and incubated with nytran filter strips containing Aa, Bg, Bi, Ec, Fn, Wr, and E. coli slots of nucleic acid. Hybridization was allowed to proceed for 3 hours at 19°C. The filters were washed with 0.09M NaCl, 50mM Tris pH 7.6, 25mM EDTA and 0.1% SDS at 50°C and radioactivity was then detected by fluorometry.
  • EXAMPLE IV Individual pyrrolidone-based hybridization solutions promote specific nucleic acid base pairing.
  • a solution composed of 40% N-cyclohexyl-2- pyrrolidone, or 40% N-hydroxymethy1-2-pyrrolidone, or 40% N- methy1-2-pyrrolidone, 50mM Tris pH 7.6, 25mM EDTA and 1% SDS was used to test the ability of 32 P-labelled specific oligonucleotide ⁇ probes (synthetic oligonucleotide probes in which the sequence is complementary to unique hypervariable regions of the 16s rRNA of the respective bacterium) to hybridize specifically to total nucleic acid from Aa, Bg, Bi, Ec, Fn, Wr, and E. coli immobilized on Nytran filters.
  • nytran slot Approximately 50 nanograms of total nucleic acid was immobilized on each nytran slot. 6.5 x 10 6 cp of 32 P-labelled probe (approximately 65 nanograms) of Aa complementary probe (Aa4B) was added to 1ml of the solution described above and incubated with nytran filter strips containing Aa, Bg, Bi, Ec, Fn, Wr, and E. coli slots of nucleic acid. Hybridization was allowed to proceed for 3 hours at 19°C. The filters were washed with 0.09M NaCl, 50mM Tris pH 7.6, 25mM EDTA and 0.1% SDS (SDS/FW) at 50°C and radioactivity was then detected by fluorometry.
  • EXAMPLE V Individual pyrrolidone-based hybridization solutions containing chaotropic agents, piperidone based hybridization solutions and caprolactam or valerolactam-based hybridization solutions promote specific nucleic acid base pairing.
  • coli immobilized on Nytran filters Approximately 50 nanograms of total nucleic acid was immobilized on each nytran slot. 6.5 x 10 6 cpm of 32 P-labelled probe (approximately 65 nanograms) of Bg complementary probe (Bg5B) was added to 1ml of the solution described above and incubated with nytran filter strips containing Aa, Bg, Bi, Ec, Fn, Wr, and E. coli slots of nucleic acid. Hybridization was allowed to proceed for 3 hours at 19°C.
  • EXAMPLE VI Specific detection of Bg bacterium in pyrrolidone-based hybridization media.
  • a hybridization media composed of 20% N-cyclohexyl- 2-pyrrolidone, and 20% N-hydroxymethyl-2-pyrrolidone, 50 mM Tris, pH 7.6, 25mM EDTA, and 2% SDS was used to lyse 1 x 10 8 cells of Aa, Bi, Ec, r, Fn, or Bg in 100 microliter volumes at 19°C Biotinylated 24-mer oligonucleotide probes complementary to conserved regions of bacterial 16s rRNA (target probes) were added to a final concentration of 100 nanograms per ml.
  • the upper aqueous phase was then decanted and nucleic acid was precipitated with 2 volumes of 100% ethanol.
  • Another 300 microliters of 1% SDS, 50mM Tris, 25mM EDTA and 0.05M NaCl was added to the organic (lower phase) and the solution was then heated to 65°C for 10 minutes and then mixed for 3 minutes at ambient temperature.
  • the nucleic acid in the upper aqueous phase was precipitated by the addition of two volumes of 100% ethanol. Nucleic acid was pelleted for 5 minutes at 10,000 rpm- at ambient temperature and the resulting pellet was solubilized. with 100 microliters of water. An aliquot was examined by gel electrophoresis.
  • Bacterial cells such as E. coli can be partially solubilized with weak chaotropic salt solutions such as guanidine-HCl which contain non-ionic detergents. Partially solubilized cells leak RNA and plasmid DNA while retaining chromosomal DNA. These observations were used to develop a method for the rapid and simple isolation of plasmid DNA from E. coli.
  • E. coli cells transfected with PBR 322 plasmid were partially solubilized with 100 microliters of 2M guanidine-HCl, 50mM Tris, 25mM EDTA and 2% Sarkosyl. The cells were incubated without agitation for 2 minutes at ambient temperature. After the incubation cells and insoluble material was pelleted at 10,000 rpm for 1 minute and the supernatant was decanted and reserved. To the supernatant 100 microliters of N-methyl-2-pyrrolidone was added followed by 500 microliters of phenol and 300 microliters of SDS-extraction buffer.
  • the solution was mixed for 3 minutes at ambient temperature and the solution was then subjected to centrifugation at 10,000 rpm for 5 minutes to force phase separation.
  • the upper aqueous phase was then decanted and nucleic acid was precipitated with 2 volumes of 100% ethanol.
  • Nucleic acid was pelleted for 5 minutes at 10,000 rpm at ambient temperature and the resulting pellet was solubilized with 100 microliters of water. An aliquot was examined by gel electrophoresis. The results indicate that relative to total extracted nucleic acid, plasmid DNA was preferentially extracted, essentially free of chromosomal DNA, rRNA and protein.
  • EXAMPLE X Extraction and immobilization of total nucleic acid from bacterial cells onto solid supports for the detection of pathogens in complex biological samples using oligonucleotide probes.
  • the solution was then subjected to centrifugation at 10,000 rpm for 5 minutes to force phase separation. 20 microliter portions of the upper aqueous phase were then slotted into Nytran filters using a slotting apparatus. The filters were then heated to 95°C for 3 hours.
  • nytran slot Approximately 50 nanograms of total nucleic acid was immobilized on each nytran slot. 6.5 x 10 6 cpm of 32 P-labelled probe (approximately 65 nanograms) of either Aa, Bg, Bi, Ek, Fn, or Wr complementary probe was added to 1ml of the pyrrolidone-based hybridization solution described above and incubated with nytran filter strips containing Aa, Bg, Bi, Ec, Fn, Wr, and E. coli slots of nucleic acid. Hybridization was allowed to proceed for 3 hours at 19°C.
  • the filters were washed with 0.09M NaCl, 50mM Tris pH 7.6, 25mM EDTA and 0.1% SDS (SDS/FW) at ambient temperature and radioactivity was then detected by fluorometry. Results indicated that,the specific probes hybridized only with their respective total nucleic acid indicating that target nucleic acid can be directly immobilized onto a solid support after extraction of chaotropic salts containing solubilized nucleic acid with lactam-based solutions.
  • EXAMPLE XI The use of pyrrolidones to isolate total nucleic acid from bacterial cells without the use of phenol.
  • Example XII One step assay to detect specific nucleic acid sequences of bacterial pathogens.
  • a pre-prepared lysis solution composed of 20% N- cyclohexyl-2-pyrrolidone, 20% N-hydroxymethyl-2-pyrrolidone, 10% N-dodecyl-2-pyrrolidone, 50 mM Tris, pH 7.6, 25 mM EDTA, and 2% SDS and containing 1 to 5 mg of 5 micron beads (silica, (Spherisorb) from Phase Sep, Deeside Ind.
  • EXAMPLE XIII Detection of Bg specific nucleotide sequences in heated GnSCN lysate using a colorimetric sandwich assay format.
  • a lysis solution composed of 3 M GnSCN, 2% Sarkosyl, 50 mM Tris, pH 7.6, 25 mM EDTA was used to lyse two sets of 1 x 10 8 cells of Bg in 100 microliter volumes at 19°C. One set was then heated in a 65°C water bath for 10 minutes while the other set remained at room temperature for the same period of time.
  • Biotinylated 24-mer oligonucleotide probes complementary to conserved regions of bacterial 16s rRNA (signal probes) were added to a final concentration of 100 nanograms per ml to both the lysate and to the 3 M GnSCN lysing solution that was to be used as the diluent.

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Abstract

L'invention concerne des nouveaux procédés de libération d'acides nucléiques à partir de cellules se trouvant dans des échantillons biologiques complexes, afin de préparer et de rendre disponible la matière d'acides nucléiques présente pour procéder à une analyse d'hybridation ou à une extraction. L'invention concerne également des nouveaux procédés d'hybridation d'acides nucléiques. L'invention concerne notamment des procédés d'isolation d'acide nucléique à partir d'un échantillon contenant un mélange biologique complexe d'acide nucléique et d'acides non nucléiques, dans lesquels l'échantillon est combiné à un milieu d'hybridation comprenant une lactame favorisant et permettant l'appariement d'acides nucléiques lorsque l'on introduit de l'acide nucléique complémentaire. La lactame représente de préférence environ 5 à environ 70 % du milieu d'hybridation et représente de préférence 2-pyrrolidone, N-éthyle-2-pyrrolidone, N-cyclohexyl-2-pyrrolidone, N-dodécyle-2-pyrrolidone, N-méthyl-2-pyrrolidone, N-hydroxyéthyl-2-pyrrolidone, N-méthyl-2-pipéridone, 2-ε-caprolactame, N-méthyl-2-caprolactame, 2-pipéridone ou N-(4-hydroxybenzyl)pyrrolidone.
PCT/US1990/004152 1989-07-24 1990-07-24 Compositions renfermant des lactames et procedes utiles pour l'hybridation d'acides nucleiques WO1991002088A1 (fr)

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Cited By (12)

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DE4224738A1 (de) * 1992-07-27 1994-02-03 Engler Blum Gabriele Verfahren zur Analyse molekularer organischer Strukturen
EP0673436A1 (fr) * 1992-07-17 1995-09-27 Aprogenex, Inc. Amplification du signal de sondes dans des etudes d'hybridation in-situ a mediation par acide nucleique
WO1996027679A1 (fr) * 1995-03-04 1996-09-12 Boehringer Mannheim Gmbh Modulation des proprietes de liaison de partenaires de liaison de l'acide nucleique
US6511805B1 (en) * 1996-03-15 2003-01-28 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
WO2010097656A1 (fr) * 2009-02-26 2010-09-02 Dako Denmark A/S Compositions et procédés pour effectuer une étape de lavage stringent dans des applications d'hybridation
US7790395B2 (en) 1996-03-15 2010-09-07 The Penn State Research Foundation Evaluation of diseases and conditions associated with translocated genes using plasma or serum DNA
JP2011521635A (ja) * 2008-05-27 2011-07-28 ダコ・デンマーク・エー／エス 新規なハイブリダイゼーションバッファーを用いた染色体異常の検出のための組成物及び方法
WO2011067678A3 (fr) * 2009-12-02 2011-08-11 Matthiesen Steen H Compositions et procédés pour réaliser des hybridations sans dénaturation
US8048629B2 (en) 1996-03-15 2011-11-01 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
EP3133166A1 (fr) 2015-08-21 2017-02-22 42 life sciences GmbH & Co. KG Composition et procede d'hybridation
EP2768974B1 (fr) * 2011-10-21 2017-07-19 Dako Denmark A/S Compositions et procédés d'hybridation
US10662465B2 (en) 2011-09-30 2020-05-26 Agilent Technologies, Inc. Hybridization compositions and methods using formamide

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US4483920A (en) * 1982-05-17 1984-11-20 Hahnemann University Immobilization of message RNA directly from cells onto filter material
EP0238332A2 (fr) * 1986-03-19 1987-09-23 Cetus Corporation Méthode d'hybridation en phase liquide et trousse de réactifs pour détecter la présence de séquences d'acides nucléiques dans des échantillons

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US4302204A (en) * 1979-07-02 1981-11-24 The Board Of Trustees Of Leland Stanford Junior University Transfer and detection of nucleic acids
US4483920A (en) * 1982-05-17 1984-11-20 Hahnemann University Immobilization of message RNA directly from cells onto filter material
EP0238332A2 (fr) * 1986-03-19 1987-09-23 Cetus Corporation Méthode d'hybridation en phase liquide et trousse de réactifs pour détecter la présence de séquences d'acides nucléiques dans des échantillons

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ANALYTICAL BIOCHEMISTRY, Vol. 163, issued 1987, THOMPSON et al., "Molecular Hybridization with RNA Probes in Concentrated Solutions of Guanidine Thiocyanate", pp. 281-291. *
PROC. NATL. ACAD. SCI. USA, issued April 1984, Vol. 81, MANSER et al., "Isolatin of Hybridomas Expressing a Specific Heavy Chain Variable Region Gene Segment by using a Screening Technique that Detects mRNA Sequences in whole Cell Lysates", pp. 2470-2474. *

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EP0673436A1 (fr) * 1992-07-17 1995-09-27 Aprogenex, Inc. Amplification du signal de sondes dans des etudes d'hybridation in-situ a mediation par acide nucleique
US5521061A (en) * 1992-07-17 1996-05-28 Aprogenex, Inc. Enhancement of probe signal in nucleic acid-mediated in-situ hybridization studies
EP0673436A4 (fr) * 1992-07-17 2002-04-24 Aprogenex Inc Amplification du signal de sondes dans des etudes d'hybridation in-situ a mediation par acide nucleique
DE4224738A1 (de) * 1992-07-27 1994-02-03 Engler Blum Gabriele Verfahren zur Analyse molekularer organischer Strukturen
US7682786B1 (en) 1995-03-04 2010-03-23 Oerum Henrik Modulation of the binding properties of nucleic acid binding partners
WO1996027679A1 (fr) * 1995-03-04 1996-09-12 Boehringer Mannheim Gmbh Modulation des proprietes de liaison de partenaires de liaison de l'acide nucleique
US7935484B2 (en) 1996-03-15 2011-05-03 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assay
US6511805B1 (en) * 1996-03-15 2003-01-28 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
US7790395B2 (en) 1996-03-15 2010-09-07 The Penn State Research Foundation Evaluation of diseases and conditions associated with translocated genes using plasma or serum DNA
US7935487B2 (en) 1996-03-15 2011-05-03 The Penn State Research Foundation Method of detecting tumor-associated DNA in plasma or serum from humans without cancer
US8361726B2 (en) 1996-03-15 2013-01-29 The Penn State Research Foundation Method of detecting tumor-associated DNA in plasma or serum with a premalignant solid tumor
US8048629B2 (en) 1996-03-15 2011-11-01 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
US11834703B2 (en) 2008-05-27 2023-12-05 Agilent Technologies, Inc. Hybridization compositions and methods
US11118214B2 (en) 2008-05-27 2021-09-14 Agilent Technologies, Inc. Hybridization compositions and methods
JP2011521635A (ja) * 2008-05-27 2011-07-28 ダコ・デンマーク・エー／エス 新規なハイブリダイゼーションバッファーを用いた染色体異常の検出のための組成物及び方法
JP2011522531A (ja) * 2008-05-27 2011-08-04 ダコ・デンマーク・エー／エス ハイブリダイゼーション組成物及び方法
EP2285979B1 (fr) * 2008-05-27 2017-01-11 Dako Denmark A/S Compositions d'hybridation et procédés
US9297035B2 (en) 2008-05-27 2016-03-29 Dako Denmark A/S Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers
WO2010097707A1 (fr) * 2009-02-26 2010-09-02 Dako Denmark A/S Compositions et procédés pour réaliser des hybridations avec dénaturation distincte de l'échantillon et de la sonde
WO2010097655A1 (fr) * 2009-02-26 2010-09-02 Dako Denmark A/S Compositions et procédés pour des applications d'hybridation d'arn
US9303287B2 (en) 2009-02-26 2016-04-05 Dako Denmark A/S Compositions and methods for RNA hybridization applications
US9309562B2 (en) 2009-02-26 2016-04-12 Dako Denmark A/S Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
US9388456B2 (en) 2009-02-26 2016-07-12 Dako Denmark A/S Compositions and methods for performing a stringent wash step in hybridization applications
WO2010097656A1 (fr) * 2009-02-26 2010-09-02 Dako Denmark A/S Compositions et procédés pour effectuer une étape de lavage stringent dans des applications d'hybridation
US11795499B2 (en) 2009-02-26 2023-10-24 Agilent Technologies, Inc. Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
CN107574230B (zh) * 2009-02-26 2022-01-11 安捷伦科技有限公司 用于进行样品和探针单独变性的杂交的组合物和方法
EP3243909A1 (fr) * 2009-02-26 2017-11-15 Dako Denmark A/S Procédés pour réaliser des hybridations avec dénaturation distincte de l'échantillon et de la sonde
CN107574230A (zh) * 2009-02-26 2018-01-12 丹麦达科有限公司 用于进行样品和探针单独变性的杂交的组合物和方法
JP2012518430A (ja) * 2009-02-26 2012-08-16 ダコ・デンマーク・エー／エス 試料及びプローブの別々の変性によるハイブリダイゼーションの実施のための組成物及び方法
US10202638B2 (en) 2009-02-27 2019-02-12 Dako Denmark A/S Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
WO2011067678A3 (fr) * 2009-12-02 2011-08-11 Matthiesen Steen H Compositions et procédés pour réaliser des hybridations sans dénaturation
US10662465B2 (en) 2011-09-30 2020-05-26 Agilent Technologies, Inc. Hybridization compositions and methods using formamide
US11118226B2 (en) 2011-10-21 2021-09-14 Agilent Technologies, Inc. Hybridization compositions and methods
EP2768974B1 (fr) * 2011-10-21 2017-07-19 Dako Denmark A/S Compositions et procédés d'hybridation
EP3133166A1 (fr) 2015-08-21 2017-02-22 42 life sciences GmbH & Co. KG Composition et procede d'hybridation
EP4276193A2 (fr) 2015-08-21 2023-11-15 42 life sciences GmbH & Co. KG Composition et procédé d'hybridation

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