WO1995019449A1 - Populations de sondes d'hybridation non adjacentes - Google Patents

Populations de sondes d'hybridation non adjacentes Download PDF

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Publication number
WO1995019449A1
WO1995019449A1 PCT/US1995/000856 US9500856W WO9519449A1 WO 1995019449 A1 WO1995019449 A1 WO 1995019449A1 US 9500856 W US9500856 W US 9500856W WO 9519449 A1 WO9519449 A1 WO 9519449A1
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Prior art keywords
probe
molecule
target
strand
moiety
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PCT/US1995/000856
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English (en)
Inventor
Nagindra Prashad
Mark Blick
William Dugald Weber
Michael Lee Cubbage
Joel Bresser
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Aprogenex, Inc.
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Priority to AU17303/95A priority Critical patent/AU1730395A/en
Publication of WO1995019449A1 publication Critical patent/WO1995019449A1/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
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • 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
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • the field of the invention is the detection of nucleic acids by using hybridization procedures.
  • nucleic acids by hybridization procedures is a valuable medical diagnostic procedure. It can, for example, be used to delect viruses, classify microorganisms, and detect genetic defects.
  • the nucleic acid moiety of a probe is frequently linked lo a delectable reporter moiety so that, after the probe as hybridized lo its target, it can be delected.
  • relatively small probes are used, each probe designed to hybridize to a particular target region in the target molecule. In such a case, in order lo double Ihc signal per probe, it can be desirable lo link a reporter moiety, such as a fluorescent moiety, al both the 3' and 5' end of each probe.
  • the reporter moieties on probes intended to hybridize to neighboring target regions could slcarically interfere with each other, preventing at least one of them from hybridizing.
  • two fluorescent moieties al the ends of probes on neighboring target regions are in loo close proximity, there could be a quenching effect as lo fluorescence.
  • the inventions are probe populations in which probes, labelled with reporter moieties at each of their ends, are designed to hybridize at spaced intervals along a target strand.
  • Related inventions are processes of using the populations.
  • DRAWING Fig. 1 Map of portion of the human papilloma virus 16 genome. Each row is in the 5' to 3' direction, from lcfl to right. Also shown arc the sequences of Ihe oligonucleotide probes used to create probe populations.
  • Aminolink 2 is 6-(trifluoroacelylamino)hcxyl-(2-cyanoethyl)-(N,N diisopropyl)- phosphoramidite and is sold by Applied Biosystems, Inc., Foster City, CA, U.S.A. It is used to create a 6-carbon linker moiety that is seven atoms long.
  • "3'-Aminomodifier C7 CPG” sold by Glen Research, Slerling, VA, U.S.A., is (1- dimethoxytrityloxy-3-fluorenylmcthoxycarbonylamino-hexan-2-melhylsuccinoyl)-Iong chain alkyl amino moiety linked to controlled pore glass. Il is used to make a 6-carbon linker moiety that is seven atoms long.
  • Aminomodificr II sold by Clontcch Laboratories, Inc., Palo Alto, CA, U.S.A., is N-Fmoc-O l -DMT-0 2 -cyanoclhoxydiisopiOpylaminophosphinyl-3-amino-l,2-propanedioI
  • amplification product is one generated by an amplification process such as PCR or 3SR, designed lo detect an analytc nucleic acid molecule.
  • the term “dye” includes any molecule or molecular moiety that can be dctccled fluorimetrically or spcctropholomelrically, especially though not necessarily in the visible range of wavelengths.
  • An "end-labelled probe” is one in which a reporter moiety is covalcnlly linked to the nucleotide or nucleoside located at the 3' or 5' end of the oligonucleotide moiety of a probe.
  • Linkage may be direel (as when an atom of the reporter moiety is directly linked lo an atom of the oligonucleotide moiety) or via a linker group, as when an atom of the reporter moiety is directly linked to an atom of a linker moiety and an atom of the oligonucleotide moiety is also directly linked to an atom of thai linker moiety.
  • LCR LCR
  • LAR LAR
  • LAS refer lo "ligation chain reaction”, “ligation amplification reaction”, and "ligalion-bascd amplification system” respectively, reactions which rely on a
  • linker moiety is a moiety thai links two other moieties ("non-linker moieties") together.
  • the "length" of a linker moiety is measured in atoms and is the number obtained by proceeding one atom at a lime along Ihe linker, starting al the alom linked to one non-linker moiety and counting until the atom linked to the other non-linker moiety, provided that the count obtained is the smallest count possible for that linker and those two nonl inker moieties.
  • a "moiety" is part of a molecule.
  • a probe molecule may consist of a dye moiety covalently linked lo a linker moiety that is covalenlly linked to an oligounculeotide moiety.
  • the phosphate group (PO 4 ) at the 5' or 3' end of a nucleic acid moiety is considered to be part of the nucleic acid moiety, independent of how il became part of the nucleic acid moiety during the procedure used to synthesize the probe molecule.
  • PCR refers to the polymerase chain reaction, an amplification process that uses oligonucleotide primers and a Taq polymerase, (see, for example, PCR protocols: a Guide to Methods and Applications. M. A. Innis et al., Eds., Academic Press, San Diego, California, 1990).
  • Q ⁇ replicase uses thai RNA bacteriophagc enzyme to effect amplification. (P. M. Lizardi et aj. Biotechnology 6, 1197 ( 1988)).
  • TAS is a transcription-based amplification system that uses oligonucleotide primers, a reverse transcriptasc, and DNA-dependenl RNA polymerase.
  • 3SR is an amplification system that uses oligonucleotide primers, a reverse transcriplase, DNA-dependenl RNA polymerase, and RNase II (J.C. Gualclli ct a[, Proc. Nail. ⁇ cad. Sci. USA, 87, 1874 (1990).
  • a “target molecule” is (he nucleic acid molecule to which a probe is intended to hybridize as a result of that probe's base sequence.
  • a target molecule may be the analyte molecule (the molecule which (he assay is designed lo delect) or a molecule, such as an amplification product, whose presence indicates the presence of an analyte molecule.
  • the invention is a process of detecting a nucleic acid target molecule, said process comprising the steps of:
  • the lcnglh of the linker not exceed 10 atoms. It is understood that the hybrid molecule comprising a probe molecule and a target molecule is formed because the nucleic acid moiety of the probe has a base sequence complementary to a base sequence of the target molecule (e.g., the adenine complementary to either uracil or thymine in the other strand, guaninc complementary to cytosinc in the other strand), ll is not necessary, however, that the entire nucleoside sequence of the probe be complementary to Ihe entire nucleoside sequence of the target.
  • a base sequence complementary to a base sequence of the target molecule e.g., the adenine complementary to either uracil or thymine in the other strand, guaninc complementary to cytosinc in the other strand
  • the invention is a process of delecting a nucleic acid target molecule, said process comprising the slcps of: 1) incubating, in a solution, a nucleic acid target molecule and a population of probe molecules, said target molecule comprised of a first strand and a second strand, said second strand complementary in base sequence lo said first strand, said probe molecules comprising base sequences that allow them lo hybridize both to discrete separated target regions on the first strands of said target molecule and discrete separated target regions on the second strand of said target molecule, and
  • Ihe invention is a population of probe molecules, wherein the base sequences of the probe molecules will allow them lo hybridize to discrete separated target regions along a target molecule, the separation between Ihe target regions being 5 lo 25 bases on average, wherein for each probe molecule, a first reporter moiety is linked cither directly or via a linker moiety indireclly lo the nucleoside at the 5' end of said probe molecule, wherein for each probe molecule, a second reporter moiety is linked cither directly or via a linker moiety indirectly to the nucleoside at the 3' end of said probe molecule, wherein the sum of the molecular weights of said first reporter moiety and said first linker moiety does not exceed 2000, and wherein the sum of the molecular weights of said second reporter moiety and said second linker moiety does not exceed 2000, said target molecule having a base sequence identical lo or complementary to a nucleic acid molecule found in a biological entity that is a cell or virus.
  • the nucleic acid moiety of a probe molecule is normally single-stranded and may be DNA, RNA.
  • the DNA or RNA may be composed of Ihe bases adenosine, uridinc, thymidine, guanine, cytosinc, or any natural or artificial chemical derivatives thereof.
  • Probes are end-labeled with a detectable reporter moiety for use in practicing the invention.
  • Typical reporter moieties are fluorescers (see Clin. Chem., 25 ⁇ :353 (1979)) chromophores; luminescers such as chcmiluminescers and bioluminescers (see Clin. Chem., 25:512 (1979)); specifically bindable ligands; and reporter moieties that are radioactive because part of their structure is a radioisotope such as 3 H, 35 S, 32 P, l25 I and C.
  • Other reporter moieties arc ones that are enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Patents Nos.
  • Biotinylated probes e.g. by PholobiotinTM labeling of probes
  • Biotinylated probes are detected after hybridization using avidin/strcptavidin, fluorescent, enzymatic or colloidal gold conjugates.
  • Nucleic acids may also be labeled with immunodetectable reporter moieties.
  • Nucleic acid probes can be used against a variety of nucleic acid targets, viral, prokaryotic, and eukaryotic.
  • the target can, for example, be a gene (e.g., oncogene), a control element (e.g., promoter, reprcssor, or enhancer), mRNA, a scc ⁇ icnce coding for ribosomal RNA, transfer RNA, or RNase P.
  • the target may be an amplification product, such as the product of PCR, 3SR, T ⁇ S, ligation chain reaction, ligation amplification reaction, ligation-based amplification system, or Q ⁇ replicase system.
  • the target may be viral DNA or RNA.
  • Viral RNA includes RNA that is genetic material, mRNA, and non- genetic material complementary to mRNA.
  • Viral DNA includes genetic material (e.g., in "DNA viruses") and the product of reverse transcriptasc (cDNA) or DNA copies thereof.
  • the scc ⁇ ienccs of many viral nucleic acids such as human immunodeficiency virus and human papilloma virus arc published and/or available through the GcnBank database.
  • a viral nucleic acid can be part of a virus, in which case the virus may or may not be inside a cell.
  • a viral nucleic acid target may not be part of a virus, but may be inside a cell.
  • the reporter moiety is a dye molecule; especially preferred is that the reporter moiety be a fluorescent dye moiety.
  • a fluorescent dye can be detected in a flow cytometer or under a microscope fitted for detection of fluorescence.
  • Preferred dyes for use as reporter moieties are fluorescent dyes that absorb light in the visible range and emit light in the visible range.
  • the target nucleic acid molecule can be a purified nucleic acid molecule or one located in a biological entity.
  • a biological entity can be a cell or a virus.
  • the cell or virus may be one that has been treated with a fixative. ⁇
  • probes bound lo the target arc delected by exposing the target-bound probe to light at a wavelength thai is absorbed by Ihe dye, and delecting the light emitted by ihe dye moiety.
  • reporter group that can be detected by a nonfluorcsccnt method is biotin, which can be detected on the basis of its ability lo bind lo a second compound, streptavidin, which in turn can be linked to enzymes such as alkaline phosphalase or horse radish peroxidase that are delectable on the basis of their ability lo react wilh a substrate.
  • Moieties that participate in chcinilumincsccnt reactions e.g., 3-aminophthalhydrazidc ("luminol"), 4-methoxy-4-(3-phosphalcphcnyl)-spiiO-(l ,2-dioxctanc-3,2'-adamantanc) disodium salt ⁇ , and moieties that react with antibodies arc also among the possibilities for reporter moieties.
  • 3-Amino-Modifier C3 CPG 3-Amino-Modifier C3 CPG, 5'-Amino-Modifier C3, or 5'-Amino-Modifier 5, or Cn-Aminomodifiers such as N- monomcthoxytrityl O-methoxydiisopropylaminophosphinyl 3-aminopropan(l)ol , and N- monomclhoxytrilyl 3-aminop ⁇ opan(l )ol (Sec Connoly, Nucleic Acids Research, vol 15, page 3131 (1987)); generally see Clonctcch's manual "DNA Modification Reagents for Use in Automated DNA Synthesis, A Users Manual (1989), including the references as to methods of synthesis on pages 16-17) ⁇ .
  • probe molecules For probes used lo delect targets in the cell nucleus (e.g., nuclear DNA) it is preferred that the probe molecules have no more than 40 nucleotides. A probe should normally be at least about 15 bases long for specific hybridization to take place. WHEN THE TARGET MOLECULE IS A PURIFIED NUCLEIC ACID
  • a purified nucleic acid is considered here to be one that has been extracted from a cell or has been synthesized in vitro in a cell-free system. Many procedures have been published for hybridizing probes to such purified nucleic acids. Generally, if the target is a DNA molecule, its strands are separated by heal or other means before the hybridization step takes place. Such hybridization can take place with the purified target nucleic acid immobilized on a solid support (e.g., nitrocellulose paper for DNA, nylon for RNA) by well-established procedures.
  • a solid support e.g., nitrocellulose paper for DNA, nylon for RNA
  • the hybridization conditions vary considerably, depending in part on the level of specificity desired. Some examples arc the Southern Blot procedure ( J. Mol. Biol., 98,
  • the hybridization assay can also be done for target molecules in biological entities.
  • the biological entity can be a cell or a virus and be cither in liquid suspension, on slides or other solid supports, in ti.ssuc culture, or in tissue sections.
  • the biological entity can come from solid tissue (e.g., nerves, muscle, heart, skin, lungs, kidneys, pancreas, spleen, lymph nodes, tcstcs, cervix, bone marrow, and brain) or cells present in membranes lining various tracts, conduits and cavities (such as the gastrointestinal tract, urinary tract, vas defcrens, uterine cavity, uterine tube, vagina, respiratory tract, nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi and lungs) or cells in an organism's fluids (e.g., urine, stomach fluid, sputum, blood and lymph fluid) or stool.
  • an organism's fluids e.g.
  • Iii situ hybridization allows the detection of RNA or DNA sequences within individual cells. With sufficiently large targets, it can detect as few as 1-5 target molecules per cell (PCT Applications WO 90/02173 and WO 90/02204). It also allows for the simultaneous detection of more than one different polynucleolidc sequence in an individual cell. It also allows detection of proteins and polynuclcotides in the same cell. Many different hybridization conditions (solvent composition, temperature, lime) are possible. The ones mentioned below arc only intended to advise the reader of some of the more preferable hybridization conditions. A person skilled in the art will know many other conditions could also be used effectively.
  • a chaotropic agent such as 50% formamide
  • a buffer such as 0.1M sodium phosphate (pll 7.4)
  • 0.1M sodium phosphate pll 7.4
  • Triton X-100 to facilitate probe entry into the cells
  • the hybridization solution is added a probe population designed to hybridize with the target nucleic acids. If the cells are to be ultimately viewed on glass slides (or other solid supports), the cells as either single cell suspensions or as tissue slices are deposited on the slides. The cells are fixed by choosing a fixative which provides the best spatial resolution of the cells and the optimal hybridization efficiency. After fixation, the support bound cells may be dehydrated and stored at room temperature or the hybridization procedure may be carried out immediately.
  • the hybridization solution containing the probe is added in an amount sufficient to cover the cells.
  • the cells are then incubated at an appropriate temperature.
  • Conditions where preferred temperatures are in the range 5 ⁇ "-55"C have been disclosed in PCT applications WO 90/02173 and WO 90/02204. However, temperatures ranging from 15°C. to 80°C. may be used.
  • the hybridization solution may include a chaotropic denaturing agent, a buffer, a pore forming agent, a hybrid stabilizing agent, and the target-specific probe molecule.
  • the chaotropic denaturing agents include formamidc, urea, thiocyanalc, guanidine, trichloroacctatc, tetramethylamine, perchloratc, and sodium iodide. Any buffer which maintains pll al least between 7.0 and 8.0 is preferred.
  • the pore forming agent is for instance, a detergent such as Brij 35, Brij 58, sodium dodccyl sulfate, CHAPSTM Triton X-100.
  • the porc-fonning agent is chosen lo facilitate probe entry through plasma, or nuclear membranes or cellular compartmental structures. For instance, 0.05% Brij 35 or 0.1 % Triton X-100 will permit probe enlry through the plasma membrane but not the nuclear membrane. Alternatively, sodium desoxycholate will allow probes to traverse the nuclear membrane.
  • nuclear membrane pore-forming agents arc avoided.
  • Such selective subccllular localization contributes to the specificity and sensitivity of the assay by eliminating probe hybridization lo complementary nuclear sequences when the target biopolymcr is located in the cytoplasm.
  • Agents other than detergents such as fixatives may serve this function.
  • Purlhcrmorc a biopolymcr probe may also be selected such that its size is sufficiently small to traverse the plasma membrane of a cell but is too large to pass through the nuclear membrane.
  • Hybrid stabilizing agents such as salts of mono- and di-valcnt cations are included in the hybridization solution lo promote formation of hydrogen bonds between complementary nucleotide sequences of the probe and its target biopolymcr.
  • nucleic acids unrelated lo the target biopolymers are added to the hybridization solution at a concentration of about 100-fold the concentration of the probe.
  • Specimens are removed after each of the above steps and analyzed by observation of cellular morphology as compared to fresh, untreated cells using a phase contrast microscope. The condition determined to maintain the cellular morphology and the spatial resolution of the various subccllular structures as close as possible to the fresh untreated cells is chosen as optimal for each step.
  • the cells Prior to nucleic acid hybridization, the cells may be reacted with antibodies in phosphate buffered saline. After hybridization one may analyze the cells for both bound antibodies and bound hybridization probes.
  • Supports which may be utilized include, but are not limited to, glass, Scotch tape (3M), nylon, Gene Screen Plus (New England Nuclear) and nitrocellulose. Most preferably glass microscope slides are used. The use of these supports and the procedures for depositing specimens thereon will be obvious to those of skill in the art. The choice of support material will depend upon the procedure for visualization of cells and the quantilalion procedure used. Some filter materials are not uniformly thick and, thus, shrinking and swelling during jn situ hybridization procedures is not uniform. In addition, some supports which aulofluoresce will interfere with the determination of low level fluorescence. Glass microscope slides are most preferable as a solid support since they have high signal-to-noisc ratios and can be treated lo better retain tissue.
  • a fixative may be a precipitating agent or a cross-linking agent used alone or in combination, and may be aqueous or non-aqueous.
  • the fixative may, for example, be selected from the group consisting of formaldehyde solutions, alcohols, salt solutions, mercuric chloride sodium chloride, sodium sulfate, potassium dichromate, potassium phosphate, ammonium bromide, calcium chloride, sodium acetate, lithium chloride, cesium acetate, calcium or magnesium acetate, potassium nitrate, potassium dichromate, sodium chromatc, potassium iodide, sodium iodate, sodium thiosulfate, picric acid, acetic acid, paraformaldehyde, sodium hydroxide, acetones, chloroform, glycerin, thymol, etc.
  • the fixative will comprise an agent which fixes the cellular constituents through a precipitating action and has Ihe following characteristics: the effect is reversible, the cellular (or viral) morphology is maintained, the antigenicily of desired cellular constituents is maintained, the nucleic acids are retained in the appropriate location in the cell, the nucleic acids are not modified in such a way thai they become unable to form double or triple stranded hybrids, and cellular constituents are not affected in such a way so as to inhibit the process of nucleic acid hybridization lo all resident target sequences.
  • Choice of fixatives and fixation procedures can affect cellular constituents and cellular morphology; such effects can be tissue specific.
  • fixatives for use in the invention include ethanol, ethanol-acetic acid, methanol, and methanol-acetone. Preferred fixatives afford high hybridization efficiency with good preservation of cellular morphology.
  • Fixatives for practicing Ihe present invention include 95% cthanol/5% acetic acid for IIL-60 and normal bone marrow cells, 75% ethanol/20% acetic acid for K562 and normal peripheral blood cells, 50% melhanol/50% acetone for fibroblast cells and normal bone marrow cells, and 10% formaldehyde/90% melhanol for cardiac muscle tissue.
  • Ethano mcthanol (3: 1 , v/v) can be used in all types of cells.
  • the fixative may contain a compound which fixes the cellular components by cross-linking these materials together, for example, glutaraldchydc or formaldehyde. While this cross-linking agent must meet all of the requirements above for the precipitating agent, it is generally more "sticky" and causes the cells and membrane components to be secured or sealed, thus, maintaining the characteristics described above.
  • cross linking agents when used are preferably less than 10% (v/v).
  • Cross-linking agents while preserving ultrastructure, often reduce hybridization efficiency; they form networks trapping nucleic acids and antigens and rendering them inaccessible to probes and antibodies. Some also covalcnlly modify nucleic acids preventing later hybrid formation. H
  • microscope slides containing cells may be stored air dried at room temperature for up lo three weeks, in cold (4"C) 70% ethanol in water for 6- 12 months, or in paraplast for up to two years. If specimens arc handled under RNase-free conditions, they can be dehydrated in graded alcohols and stored for at least 5 months at room temperature.
  • Reagents can be purchased from any of a variety of sources including Aldrich Chemical Co., Milwaukee, Wisconsin, Sigma Chemical Co., Si. Louis, Missouri, Molecular Probes, Inc., Eugene, Oregon, Clontcch, Palo Alto, California, Kodak, Rochester, NY, and Spectrum Chemical Manufacturing Corp., Gardcnea, California.
  • cells either as single cell suspensions or as tissue slices may be deposited on solid supports such as glass slides.
  • cells arc placed inlo a single cell suspension of about 10 s - 10° cells per ml.
  • the cells are fixed by choosing a fixative which provides the best spatial resolution of the cells and the optimal hybridization efficiency.
  • the hybridization is then carried out in the same solution which effects fixation.
  • This solution contains both a fixative and a chaotropic agent such as formamide.
  • a hybrid stabilizing agent such as concentrated lithium chloride or ammonium acetate solution, a buffer, low molecular weight DNA and/or ribosomal RNA (sized to 50 bases) lo diminish non-specific binding, and a pore forming agent lo facilitate probe entry into the cells.
  • Nucleasc inhibitors such as vanadyl ribonuclcoside complexes may also be included.
  • a probe or probes
  • lo hybridize with a target polynucleotide.
  • the one-step procedure is a means of carrying out the fixation, prehybridizalion, hybridization and detection steps normally associated witli in situ hybridization procedures all in one step.
  • a convenient tcmperalure may be used to carry out the hybridization reaction.
  • this provides a hybridization assay which can be accomplished with viable or non-viable cells in solution. In either case, the assay is rapid and sensitive.
  • the one-step hybridization procedure is carried out utilizing a single hybridization solution which also fixes the cells. This fixation is accomplished in the same solution and along with the hybridization reaction.
  • the fixative may be selected from the group consisting of any precipitating agent or cross-linking agent used alone or in combination, and may be aqueous or non-aqueous.
  • Tissue samples are broken apart by physical, chemical or enzymatic means into single cell suspension.
  • Cells arc placed into a PBS solution (maintained to cellular osmolality with bovine serum albumin (BSA) al a concentration of 10 s lo 10 ft cells per ml.
  • BSA bovine serum albumin
  • Cells in suspension may be fixed and processed at a later lime, fixed and processed immediately, or not fixed and processed in the in situ hybridization system of the present invention.
  • a single solution is added lo the cells/tissues (hereafter referred lo as the specimen).
  • This solution contains the following: a mild fixative, a chaotropc, a nucleic acid probe (RNA or DNA probe which is prelabcled) and/or antibody probe, salts, detergents, buffers, and blocking agents.
  • the incubation in this solution can be carried out al 55"C for 20 minutes as well as other conditions such as those in the Example below.
  • the fixative is preferably one that has been found lo be optimal for the particular cell type being assayed (eg., there is one optimal fixative for bone marrow and peripheral blood even though this "tissue.” contains numerous distinct cell types).
  • the fixative is usually a combination of precipitating fixatives (such as alcohols) and cross-linking fixatives (such as aldehydes), with the concentration of the cross-linking fixatives kept very low (less than 10%). Frequently, the solution contains 10-40% ethanol, and 5% formalin.
  • concentration and type of precipitating agent and crosslinking agent may be varied depending upon the probe and the stringency requirements of the probe, as well as the desired temperature of hybridization. Typical useful precipitating and cross-linking agents are specified in PCT applications WO 90/02173 and WO 90/02204.
  • the hybridization cocktail contains a denaturing agent, usually formainide at about 30% (v/v), but other chaotropic agents such as Nal, urea, etc. may also be used. Furthermore, several precipitating and/or cross-linking fixatives also have mild denaturing properties; these properties can be used in conjunction with the primary dcnalurant in cither an additive or synergistic fashion.
  • the hybridization cocktail may be constructed to preferentially allow only the formation of RNA-RN ⁇ or RNA-DNA hybrids. This is accomplished by adjusting the concentration of the denaturing agents along with the concentration of sails (primarily monovalcnt cations of the Group I series of metals along with the ammonium ion) and along with the temperature of hybridization which is used.
  • probes This allows for the selective hybridization of probe to either cellular RNA or DNA or both RNA and DNA simultaneously with distinct probes. This further allows the probes to be supplied in a premixed solution which presents the optimal conditions for generating a signal and minimizing noise while simultaneously optimally "fixing" the morphology of the cells/tissues.
  • probes against both strands of a double-stranded target can be used without probes hybridizing to each other, provided that a probe that hybridizes to one strand is complementary in base sequence to no more than 15 bases of a probe that hybridizes to the other strand, a situation that can be achieved by having the target base sequences along one strand out-of-phase as to the target sequences along the other strand.
  • kits for detecting a target nucleic acid molecule in a biological entity comprising a probe population described herein and one or more reagents (selected from the group, a fixative and a chaotropic agent) for use in a solution for reacting said probe population with said biological entity so that a hybrid molecule can form between a molecule of the probe population and a nucleic acid target molecule in the biological entity.
  • a kit could include a solution containing a fixation/hybridization cocktail and one or more probe populations of this invention.
  • This solution could, for example, contain 15-40% ethanol, 25-40% formamide, 0-10% formaldehyde, 0.1-1.5 M LiCl , 0.05-0.5 M Tris-acetale (pll 7-8), 0.05%-0.15% Triton X-100, 20 ug/ml-200 ug/ml of a non-specific nucleic acid which docs not react with Ihe probe(s), and 0.1 ug/ml to 10 ug/ml of single stranded probes directly labeled with a reporter molecule. More specifically, for example, this solution could contain 30% ethanol, 30% formamide, 5% formaldehyde, 0.8M LiCl , 0.
  • the kil could include concentrated stock solution(s) lo be diluted sufficiently to form the solutions needed for hybridization. Additionally, it could include any mechanical components which may be necessary or useful to practice the present invention such as a solid support (e.g. a microscope slide), an apparatus lo affix cells to said support, or a device to assist with any incubations or washings of the specimens, and/or a photographic film or emulsion with which lo record results of assays carried out with the present invention. Normally, the kil would include instructions on how to carry out the hybridization.
  • Fluorescent measurements can be made using a fluorescent microscope such as an Olympus BH10 microscope wilh fluorescent capabilities. Fluorescent measurements can also be made on a flow cytometer, such as a FACSTARTM made by Becton Dickinson. EXAMPLES
  • DNA synthesizer To create oligonucleotidcs wilh a dye linkers al the 5' end only, a DNA synthesizer is used. DNA synthesizers and literature describing the required phosphoroamidilc chemistry can be obtained from various sources including Applied Biosyslcms, Inc.
  • ABSOR Advanced Bioscicnces, Inc., Madison, MN, Cruachem, Dulles, VA, Biogenex, San Ramon, CA, Milligen, Watertown, MA, and Pharmacia, Piscataway, NJ (Sec, for example, ABI DN ⁇ /RNA Synthesizers User's Manual, Models 392,394, Doc. Rev. A., Applied Biosyslcms, August, 1992; ABI User Bulletin, Model 370, Issues 11, Jan. JO, 1989; Nature 32J. 674 (1986)).
  • the automated synthesizer is used in Ihe same way that it is used to normally (no dye linkers) create an oligonucleotide by standard phosphoramidile chemistry except thai a linker is added instead of a nucleoside in the last synthesis step. More specifically the steps are as follows: 1) A DNA Synthesizer is equipped wilh a support made, for example, of controlled-pore glass (CPG) or polystyrene; the CPG is attached via a linker to a nucleoside whose 5' hydroxyl is blocked with dimcthoxylrilyl (DMT). That nucleoside will be the 3' nucleoside of the complete oligonucleotide.
  • CPG controlled-pore glass
  • DMT dimcthoxylrilyl
  • oligonucleotide is then detrytilated and ils resulting 5' hydroxyl group is reacted with the phosphoramidile compound, Aminomodifier II (Clonetcch Laboratories, Inc.), instead of another nucleoside. 5) Continuing wilh the DNA synthesizer as in a normal oligonucleotide cycle, all Ihe protecting groups (e.g., the li fluorocarbonyl group on the Aminomodifier 11, and the groups protecting the phosphate groups) arc removed and the oligonucleotide is cleaved from the solid support linker.
  • Ihe protecting groups e.g., the li fluorocarbonyl group on the Aminomodifier 11, and the groups protecting the phosphate groups
  • the cleavage creates an oligonucleotide wilh a a 3' hydroxyl group and a 5' primary amine linker attached lo an oxygen atom of the 5' phosphate group of the 5' nucleoside.
  • the primary amine group of Ihe aminoethyl linker is available for reaction with a fluorescent dye.
  • a DNA synthesizer is used to create oligonuclcotidcs wilh dye linkers at the 5' and 3' ends.
  • the automated synthesizer is used in the same way is used to normally (no dye linkers) create an oligonucleotide by standard phosphoramidile chemistry, except that special linkers link the oligonucleotide lo the column and a linker is added instead of an nucleoside as the last base. More specifically the slcps are as follows:
  • a DNA Synthesizer is equipped wilh ( l-dimelhoxylrityloxy-3- fluorenylmethoxycarbonylamino-propan-2-succinoyl)-long chain alkylamino-CPG ("3'-
  • Amino-Modifier C7 CPG sold by Glen Research (Sterling, VA) is used.
  • the 3'-Amino Modifier C7 CPG is detrilylated so as to create an hydroxyl group and that group is reacted with a phosphoramidite nucleoside.
  • nucleosides are added by standard DNA Synthesizer phosphoramidite chemistry.
  • phosphoramidite compound Aminolink 2 reacts with the oligonuclcotide's 5' hydroxyl group in the same manner as a protected phosphoramidile nucleoside would react wilh that hydroxyl during normal oligonucleotide synthesis by the DNA synthesizer.
  • the phosphate group created by that reaction is protected by a methyl group.
  • Example 2 The procedure of Example 2 is followed except that Aminomodifier II (from Clonetech Laboratories, Inc.) is used instead of Aminolink 2. Groups eliminated by the deprotection step include the fluorenylmelhoxy carbomyl ("Fmoc") group contributed by the 3'-Amino-modifier C7 CPG.
  • Aminomodifier II from Clonetech Laboratories, Inc.
  • Aminolink 2 Groups eliminated by the deprotection step include the fluorenylmelhoxy carbomyl (“Fmoc") group contributed by the 3'-Amino-modifier C7 CPG.
  • TAMRA is 5- (and -6) -carboxytetramethylrhodamine succinimidyl ester; sold by Molecular Probes, Cat No. 1171)
  • step 8 Resuspend the pellet in 50 ⁇ l water and pass through a Sephadcx G-50 exclusion column. Collect the first colored band (DNA) and dry using a vacuum concentrator.
  • the cells used were CASKI cells (American Type Culture Collection # CRL1550) containing 400-500 copies of the IIPV 16 genome inlcgraled into Ihc cellular genome and, as a negative control, C-33A cells (ATCC #HTB31). Cells were grown to confluence in 5% C0 , then rinsed in IX PBS. To the cells were added 0.25% trypsin in 0.02 M EDT ⁇ . They were incubated at 37°C for 5 min, then gently tapped to dislodge cells. They were then washed in media and then spun by cytospin for about 7 min at 700 rpm onto clean glass slides and left to air dry. To the dried cells was added 20 ul of cthanohmelhanol (3: 1 ). They were then allowed to dry.
  • Hybridization was done by incubating the cells on slides in 20 ul of a hybridization cocktail al 42 °C for 5 min, Ihen 90 "C for 5 min, then 42 "C for 20 min after which the cells were washed once with wash solution A and five times wilh wash solution 13 before being mounted in mounting solution.
  • the hybridization solution was made according lo the following formula:
  • wash solution A had the following composition: 0.4 M guanidium isothiocvanale, 0.1 % Triton X- 100, 0.1 x SSC in deionized water.
  • Wash solution B had the following composition: 0.1 % Triton X-100, 0.1 x SSC in deionized water.
  • Mounting solution was 0.1% 1 ,4 diphenylamine (anlifade) in 50% glycer ⁇ l (v/v) and nuclear stain Hoechst (#33258; 1 ug/ml).
  • PEG is polyethylene glycol.
  • SSC is 0.15 sodium citrate, 0.015 M sodium citrate.
  • Ficoll/PVP is 5 g of Ficoll type 400 (polysucrose 400,000 molecular weight) plus 5 g of PVP (polyvinylpyrrolidone) diluted to a total volume of 100 ml with water.
  • Sodium phosphate buffer is ph 6.8.
  • the fluorescent signal was measured by a fluorescent microscope and compared to each other by intensity. An Olympus BII-2 microscope was used.
  • Fig. 1 is a sequence map of a portion of the "sense strand" human papilloma virus genome.
  • the top strand is the sense strand; the bottom strand is the anli-sense strand.
  • Base sequences in each strand were used to define the sequences of a series of oligonucleotides 25 bases in length ("25-mcrs") and separated by five bases along that strand.
  • Two probe populations, A and B were constructed such that each had all the probes whose sequences were defined by the sense strand (therefore those probes were capable of hybridizing to the anti-sense strand).
  • Two probe populations, C and D were constructed such that each had all the probes whose sequences were defined by the anti- sense strand.
  • the probes of populations A and B had probes equivalent to discrete regions 701- 71 1 of the sense strand with their Nucleotide Sequence ID No. as follows:
  • SEQ ID NO: 13 is the sequence of the entire sense strand in Fig. 1.
  • the probes of populations C and D had probes equivalent to discrete regions 600- 1 1 of the antisense strand with their Nucleotide Sequence ID No. as follows:
  • 61 1 are discrete target regions.
  • the discrete target regions form an overlapping pattern in
  • first strand is complementary in base sequence lo a ten-base sequence at an end of a
  • Target sequences at the ends of the pattern are discrete target region on the other strand.
  • Every probe in populations A and C had one tetramethylrhodamine moiety linked to its 5' end -nucleoside, but no tetramethylrhodamine or other reporter moiety linked to its 3'-end nucleoside.
  • Every probe in populations B and D had a tetramethylrhodamine moiety linked to the nucleoside at its 3' end and a tetramethylrhodamine entity linked to the nucleoside at its 5' end.
  • the probes in Population A and C were made using the procedure of Examples 1 and 4, thereby using Aminomodifier II to create the moiety linking the tetramethylrhodamine lo the 5' nucleoside.
  • the probes in population B and D were made using the methods of Examples 3 and 4, thereby using AminoModificr II to create the ifioicly linking the tetramethylrhodamine to the 5' nucleoside and using 3'-Amino-Modifier C7 CPG to create the moiety linking the tetramethylrhodamine lo the 3' nucleoside.
  • results show that the signal observed when both ends of a probe are labeled probe is about twice as great as that observed when only one end is labeled.
  • results also show that, when both ends of a probe are labeled, the signal obtained wilh a probe population that has probes for both target strands is about twice that obtained wilh a population that has probes for only a single strand.
  • ADDRESSEE Elraan, Wilf & Fried
  • B STREET: 20 West Third Street
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL N
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL N
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL N
  • SEQUENCE DESCRIPTION SEQ ID NO: 15:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL N
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Populations de sondes dans lesquelles les sondes, marquées avec des fractions rapporteuses à chacune de leurs extrémités, sont prévues pour s'hybrider à des intervalles espacés le long d'un brin cible. Cette invention concerne également des procédés d'utilisation de ces populations.
PCT/US1995/000856 1994-01-14 1995-01-13 Populations de sondes d'hybridation non adjacentes WO1995019449A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0731173A2 (fr) * 1995-03-10 1996-09-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Procédé pour la détection directe d'un nombre réduit de oligonucléotides
EP1002877A2 (fr) * 1998-11-09 2000-05-24 Sanko Junyaku Co., Ltd. Procédé pour l'amplification génique
WO2000031302A1 (fr) * 1998-11-25 2000-06-02 Isis Pharmaceuticals, Inc. Synthese binaire in situ de molecules biologiquement efficaces
US6492111B1 (en) * 1998-11-25 2002-12-10 Isis Pharmaceuticals, Inc. In situ binary synthesis of biologically effective molecules
WO2012152708A1 (fr) * 2011-05-06 2012-11-15 Qiagen Gmbh Oligonucléotides comprenant un marqueur lié par l'intermédiaire d'une séquence de liaison

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4983511A (en) * 1989-01-09 1991-01-08 Olin Corporation Method and kit for detecting live microorganisms in chlorine- or bromine-treated water
US5273882A (en) * 1985-06-13 1993-12-28 Amgen Method and kit for performing nucleic acid hybridization assays

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5273882A (en) * 1985-06-13 1993-12-28 Amgen Method and kit for performing nucleic acid hybridization assays
US4983511A (en) * 1989-01-09 1991-01-08 Olin Corporation Method and kit for detecting live microorganisms in chlorine- or bromine-treated water

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 29, issued 1990, J.P. COOPER et al., "Analysis of Fluorescence Energy Transfer in Duplex and Branched DNA Molecules", pages 9261-9268. *
STRATAGENE CATALOG, issued 1988, page 39. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0731173A2 (fr) * 1995-03-10 1996-09-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Procédé pour la détection directe d'un nombre réduit de oligonucléotides
EP0731173A3 (fr) * 1995-03-10 2000-02-23 Evotec BioSystems AG Procédé pour la détection directe d'un nombre réduit de oligonucléotides
JP2009039131A (ja) * 1995-03-10 2009-02-26 Evotec Biosystems Gmbh 少数のヌクレイン酸鎖を直接証明する方法
JP4542182B2 (ja) * 1995-03-10 2010-09-08 エボテク バイオシステム ゲーエムベーハー 少数のヌクレイン酸鎖を直接証明する方法
EP1002877A2 (fr) * 1998-11-09 2000-05-24 Sanko Junyaku Co., Ltd. Procédé pour l'amplification génique
EP1002877A3 (fr) * 1998-11-09 2002-02-20 Sanko Junyaku Co., Ltd. Procédé pour l'amplification génique
WO2000031302A1 (fr) * 1998-11-25 2000-06-02 Isis Pharmaceuticals, Inc. Synthese binaire in situ de molecules biologiquement efficaces
US6492111B1 (en) * 1998-11-25 2002-12-10 Isis Pharmaceuticals, Inc. In situ binary synthesis of biologically effective molecules
WO2012152708A1 (fr) * 2011-05-06 2012-11-15 Qiagen Gmbh Oligonucléotides comprenant un marqueur lié par l'intermédiaire d'une séquence de liaison
CN103502474A (zh) * 2011-05-06 2014-01-08 凯杰有限公司 包含经由连接基连结的标记的寡核苷酸
US9150912B2 (en) 2011-05-06 2015-10-06 Qiagen Gmbh Oligonucleotides comprising a label associated through a linker

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