EP0462221A1 - Hybridation in situ en suspension pour la detection ou la separation de cellules - Google Patents

Hybridation in situ en suspension pour la detection ou la separation de cellules

Info

Publication number
EP0462221A1
EP0462221A1 EP90905309A EP90905309A EP0462221A1 EP 0462221 A1 EP0462221 A1 EP 0462221A1 EP 90905309 A EP90905309 A EP 90905309A EP 90905309 A EP90905309 A EP 90905309A EP 0462221 A1 EP0462221 A1 EP 0462221A1
Authority
EP
European Patent Office
Prior art keywords
probe
cells
cell
suspended
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP90905309A
Other languages
German (de)
English (en)
Inventor
Jo Anne H. Kerschner
Edward G. Jablonski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngene Inc
Original Assignee
Molecular Biosystems Inc
Syngene Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Molecular Biosystems Inc, Syngene Inc filed Critical Molecular Biosystems Inc
Publication of EP0462221A1 publication Critical patent/EP0462221A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • C12Q1/6841—In situ hybridisation

Definitions

  • This invention relates generally to nucleic acids and more specifically to techniques for hybridizing and detecting nucleic acid sequences within a cell while the cell is in suspension.
  • Complementary strands of nucleic acid such as DNA or RNA can form a double-stranded molecule linked by complementary bases in a process known as hybridization.
  • Hybrids may be formed between any two complementary strands, so hybrids of DNA:DNA are possible, as are DNA:RNA and RNA:RNA.
  • the reaction is reversible under particular temperature and salt concentration conditions.
  • nucleic acids are first isolated and then permitted to hybridize with particular labelled nucleotide sequences termed probes. While such nucleic acid hybridization is well known in the art, methods for hybridization of nucleic acid sequences within cells in suspension have not been available.
  • in situ hybridization has been used to detect DNA sequences within suspended nuclei removed from cells, Trask, B., et al., Science, 230:1401-03, (1985), Trask, B. , et al.. Human Genetics, 78:251-59 (1988), Van der Engh, G.J. and Trask, B.J. (1988), U.S. Patent No. 4,770,992.
  • Trask, et al. cross-linked nuclei, isolated from cells, with dimethylsuberimidate to prevent disintegration of the nuclei during denaturation and hybridization.
  • AAF N-acetoxy-2-acetylaminofluorene
  • AAIF iodinated analog
  • biotin-labelled chromosomal and cloned DNA probes were hybridized to their respective targets and the probes detected with anti-AAF or anti-AAIF antibodies and a secondary rhodamine-labelled antibody (for AAF- or AAIF-labelled probes) or fluorescein-avidin-DSC (for biotin-labelled probes) by flow cytometry.
  • Flow cytometry is a technique for rapidly sorting individual cells, microorganisms and cell organelles into groups based on observable differences. Briefly summarized, the technique involves passing a stream of material to be measured (cells, lysed cells or other material) in single file past a light source where the particular property is detected, usually based on the reaction of the light to the materials. A computer then directs the separation of the material as desired. This technique is very rapid and allows separation of from 10 4 to 10 6 cells per minute.
  • Fluorochromes are most often used to label the cellular component of interest before passing the cell sample past the light source.
  • a variety of fluorochromes have been used to nonspecifically stain DNA, RNA and proteins for cytometric analysis.
  • Immunological techniques have been used to detect cell surface markers and cell interior markers specifically with fluorescent antibodies.
  • Nonfluorescent dyes can also be utilized in this system.
  • Cells can absorb light as they pass through a focused light beam, and light transmitted by cells stained with nonfluorescent dyes can be quantitated and related to analyte concentration. This technique can help measure the expression of various target molecules in cells.
  • a suinmary of flow cytometry as a technique is provided in Muirhead, K.A. , et al., Biotechnology, 3:337- 56 (1985).
  • the present invention provides a technique for the hybridization of nucleotide probes to nucleic acids within a cell while the cell remains in suspension.
  • the technique comprises fixing the suspended cell, hybridizing the nucleic acid within the cell with an oligonucleotide probe and detecting the hybridized probe.
  • the nucleic acid can be denaturated prior to hybridization and endogenous enzymes can be ablated.
  • the invention also provides a technique for categorizing cells by flow cytometry based on differences in target nucleic acid sequence. Additional objects and advantages of the invention will be apparent to those skilled in the art upon reading the specification and claims.
  • a cell suspended in fluid having a hybridized nucleotide probe therein is also provided as well as a kit for detecting a specific nucleotide sequence in a cell suspended in a fluid.
  • the kit comprises a means for fixing the suspended cell and a nucleotide probe complementary to a nucleotide sequence within the suspended cell.
  • the kit can also include a means for detecting the hybridized probe.
  • a method for detecting a specific nucleotide sequence in the nucleic acid in a cell suspended in a fluid comprises (l) fixing the suspended cell in such a way as to permit a nucleotide probe to penetrate without disrupting the suspended cell, (2) providing a nucleotide probe complementary to the specific nucleotide sequence within the suspended cell under conditions permitting hybridization, and (3) detecting hybridized probe within the cell, whereby hybridized probe indicates the presence of the specific nucleotide sequence.
  • the method can include the further step of denaturing the nucleic acid before hybridizing with a nucleotide probe or ablating endogenous enzymes.
  • probe or nucleotide probe refers to a labelled nucleotide sequence.
  • the nucleotides can be deoxyribonucleotides or ribonucleotides.
  • the number of nucleotides present in the probe is variable, depending on the application. Preferably, the range is between 10 and 2000 nucleotides, more preferably between 15 and 50, most preferably 20 to 25.
  • oligonucleotide refers to nucleotide sequences less than 200 residues in length.
  • Various labels are well known in the art and include fluorophores, enzymes, and radioactive isotopes, haptens and other ligands.
  • probes are described, for example, in PCT Publication No. WO 84/03285.
  • the probe can be a single selected homogeneous sequence or can be a mixture of various selected sequences.
  • a mixture can be used, for example, to increase the sensitivity of the assay or to permit detection even where a portion of the nucleic acid in the target cell is variant, as a result, for example, of genetic alteration.
  • the present invention is useful for detecting a target nucleic acid sequence in a single cell in suspension.
  • the technique may be achieved using automatic flow analysis, for example, flow cytometry.
  • the technique involves manufacturing oligonucleotides by methods known in the art. See, for example. United States Patent No. 4,500,707 Matteucci and Caruthers, (1981), J. Am. Chem. Soc., 103:3185-3191, which is incorporated herein by reference. These oligonucleotides are then labelled with enzymes, fluorophores or ligands (molecules with high affinity for other molecules) , also by means known in the art. See, for example Jablonski, E.G., et al.
  • oligonucleotides may be used as probes for hybridization with DNA or RNA in the nucleus or in the cytoplasm of cells. The presence of hybridized cellular DNA or RNA is then established by detecting the label on the probe.
  • the ji situ hybridization assay set forth here may be used in tandem with flow cytometry to assist for example in the diagnosis of various agents that infect or invade cells (viruses, chlamydia, certain bacteria and certain parasites) and to diagnose cancer and genetic diseases.
  • Detection by microscopic analysis can be used in place of flow cytometry, but flow cytometry is preferred.
  • the technique of the present invention may be combined with flow cytometry to form an automated, rapid detection or quantitation system for use in hospitals and research laboratories.
  • the technique of the present invention may also be used separately and the labelled cells may be detected in a subsequent step, if desired.
  • a technique of the present invention may be briefly summarized as follows: (1) oligonucleotide probes are synthesized and labelled with enzymes, fluorophores, ligands or other detectable labels; (2) the probes are then hybridized with DNA or RNA in a fixed suspended cell, using the methods set out below; (3) the labels in the cells are detected using known optical techniques; and (4) the cells are separated, if desired.
  • synthetic probes preferably DNA are prepared using conventional techniques.
  • the probes are designed to be complementary to particular nucleotide sequences, termed target sequences of nucleic acids within the fixed cell.
  • the selection of nucleotide sequence of the probe is within the skill and choice of those in the art.
  • long chain probes greater than 200 nucleotides
  • synthetic oligonucleotide probes (10 to 200 nucleotides, preferably 15 to 50 nucleotides) are preferred. Since higher concentrations of oligonucleotide probes can be used in the technique of the invention, maximum hybridization can usually occur within a short time, for example, ten minutes or less.
  • oligonucleotides may be labelled directly, while long probes must typically be labelled indirectly, adding complexity and expense to the process but without providing significant additional reliability.
  • a directly labelled probe is one where the label to be detected is directly attached to the probe.
  • An indirectly labelled probe is one which has attached to it a moiety through which the label to be detected is subsequently attached. Additionally, short probes can more easily permeate cells than can longer probes.
  • labels include enzymes, fluorophores and ligands. Other labels are available and are well known in the art. Enzyme labels include alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, glucose oxidase and luciferase. Fluorophores include fluorescein and Texas Red.
  • Ligand labels include biotin, digoxigenin, AAF and AAIF.
  • Protected linker arm nucleoside 3'-phosphoramidite was prepared by the method of Ruth, J. DNA, 3:123 (1984) .
  • linker arm monomer was incorporated directly into automated oligonucleotide synthesis employing an Applied source
  • the preferred conditions for nucleic acid denaturation is a combination of heat 50 e C to 100°C, preferably 65°-70°C, and a formamide concentration of 40% to 100%, preferably about 70% ultrapure: 30% low salt buffer) .
  • the hybridization technique is useful for in situ hybridization within cells in suspension. Before hybridization can occur, the cell must be fixed. Fixation is the chemical preservation of cells or tissue so that the structure will be minimally altered from the normal state. There are generally two types of fixatives, crosslinking (for example, parafor aldehyde, glutaraldehyde) and precipitating (alcohols) .
  • fixatives for example, parafor aldehyde, glutaraldehyde
  • precipitating alcohols
  • fixation with paraformaldehyde is preferred, although other crosslinking fixatives will achieve effective results.
  • the preferred amount of paraformaldehyde is between 0.5% and 5%. Precipitating fixatives are less desirable, as cell morphology is not well maintained and cells tend to clump.
  • nucleic acid within the cells can be denatured.
  • Denaturing agents or conditions for nucleic acid include formamide, glyoxal, heat (90°C to 100 ⁇ C, preferably 0 to 0.3M salt concentration), acid or base (DNA only) treatments. Denaturation with a combination of heat (>65 ⁇ C) and formamide (both DNA and RNA denaturation) is preferred. Where the secondary structure of the nucleic acid does not provide a barrier to hybridization, for example where the nucleic acid is shorter, single stranded RNA or where the probe is short and recognizes an exposed sequence, denaturation is not necessarily required.
  • probe in hybridization buffer is added immediately and hybridization commences.
  • Conditions for hybridization depend on probe base content. Hybridization can occur in the presence or absence of formamide ( ⁇ 40%) and in a temperature range of ambient temperature (23 ⁇ C) to 75°C for alkaline phosphatase probes; the temperature and formamide concentration are experimentally determined. Washing of unreacted or loosely bound probe from cells occurs at a temperature and salt concentration which are likewise experimentally determined (23-75°C, ⁇ 0.3M) .
  • Detection of alkaline phosphatase labelled probe hybridized to its complement in cells occurs preferably with the addition of NBT and BCIP substrates, although other substrates (especially substrates which give insoluble products) can also be utilized. A red to purple product is desired.
  • In situ hybridization of cells, accomplished in suspension is well adapted to automation and thus is highly desirable for applications where a complete automated system is preferable, for example, for clinical diagnostic purposes.
  • Cytomegalovirus (CMV)-infected and uninfected human fetal foreskin fibroblast (HFFF) cells were treated with 0.5% paraformaldehyde in phosphate-buffered saline (PBS) , a common buffer, for one minute at 60°C. Treating for ten minutes at ambient temperature (23°C) was an acceptable alternative.
  • PBS phosphate-buffered saline
  • the fixed cells were suspended for two minutes at room temperature with 0.2N HC1.
  • the nucleic acids within the suspended cells were denatured with a mixture of 70% formamide (ultrapure) and 30% 2X SSC (0.3M sodium chloride, 0.03M sodium citrate, pH 7.0) for 10 minutes at 70-C.
  • Hybridization buffer contains 5X SSC (0.75 sodium chloride, 0.075M sodium citrate, pH 7.0), combined with 0.5% bovine serum albumin (BSA, Fraction V). Alternatively, the cells and probe were mixed with 30% formamide in 70% hybridization buffer at 37°C for ten minutes.
  • CMV sequences are available from GenBank, (IntelliGenetics, Mountain View, CA) Accession Nos. K01090, M10063, M11911) . The following nucleotide sequences were used as probes:
  • Substrate was added to cells so that the labelling enzyme was detected.
  • BCIP 3-indolylphosphate
  • AP alkaline phosphatase buffer
  • 0.1M Tris-HCl, pH 8.5; 0.1M NaCl, 0.05M MgCl 2 ; and O.l ⁇ tM ZnCl 2 was pre-warmed to 37°C and added to the cells. Incubation took place for 1.0 hour, during which time the enzyme label converted the substrate to an insoluble purple dye.
  • the cells were rinsed with distilled water and were ready for analysis and/or separation by acceptable methods, including cell cytometry. Cells positive for virus turned an opaque blue-purple, while uninfected cells remained colorless.
  • HIV Human immunodeficiency virus
  • CEM cells peripheral blood mononuclear cells
  • the fixed cells were suspended for two minutes at room temperature in 0.2N HC1.
  • the nucleotides in the suspended cells were denatured with a mixture of 70% formamide (ultrapure) and 30% 2X SSC for 10 minutes at 70°C.
  • the cells were immediately hybridized by placing them in hybridization buffer with a mixture of twenty 2.5 nM alkaline phosphatase-labelled probes (20-24 bases) specific for HIV for ten minutes at 55"C. HIV sequences are available from GenBank, Accession number K03455. Labelling was as described in Example I. Excess probe was washed out of the cells three times, with IX SSC at 45°C.
  • Substrate was added to cells so that the labelling enzyme was detected.
  • a 1 ml solution of 0.33 mg/ml nitro blue tetrazolium (NBT) and 0.17 mg/ml 5-bromo-4-chloro- 3-indolylphosphate (BCIP) in alkaline phosphatase (AP) buffer was pre-warmed to 37°C and added to the cells. Incubation took place for 0.5 hours, during which time the enzyme label converted the substrate to an insoluble purple dye.
  • the cells were rinsed with distilled water and were ready for analysis and/or separation by acceptable methods, including cell cytometry. Cells positive for virus turned an opaque blue-purple, while uninfected cells remained colorless.
  • Herpes simplex virus type 1 (HSV)-infected and uninfected HEp-2 cells were treated with 0.5% paraformaldehyde in PBS for one minute at 60°C. The fixed cells were then suspended for two minutes at room temperature in 0.2N HC1. The cells were further treated, in suspension, with 0.01% hydrogen peroxide in methanol for 20 minutes at ambient temperature.
  • HSV Herpes simplex virus type 1
  • the nucleic acids in the cells were denatured with a mixture of 70% formamide (ultrapure) and 30% 2X SSP (0.36M NaCl, 20 mM NaH 2 P0 4 , pH 7.0) 10 minutes at 70°C.
  • the cells were immediately hybridized by placing them in "hybridization buffer II" with 5 nM horseradish peroxidase-labelled 22 base probe specific for HSV1 and HSV2 for ten minutes at 50°C.
  • the HSV sequence is available from GenBank, Accession No. J02224.
  • Hybridization buffer II contained 5X SSP (0.9M NaCl; 50 mM NaH 2 P0 4 , pH 7.0) and 0.5% BSA (Fraction V). Excess probe was washed out of the cells three times, with IX SSP (0.18M NaCl; lOmM NaH 2 P0 4 , pH 7.0) at 50°C.
  • Substrate was then added to the cells so that the labelling enzyme could be detected.
  • a 1 ml solution of 0.5 mg/ml 0-dianisidine dihydrochloride in HRP buffer (0.1M imidazole, 0.1M NaCl, pH 7.4 with 0.001% hydrogen peroxide) was added to the cells. Incubation took place for 1.0 hour at ambient temperature, during which time the enzyme label converted the substrate to an orange dye.
  • the cells were rinsed with distilled water and were ready for analysis and/or separation by acceptable methods, including cell cytometry. Cells positive for virus turned orange, while uninfected cells remained colorless.
  • Herpes simplex virus (HSV)-infected and uninfected HEp-2 cells are treated with 0.5% paraformaldehyde in PBS. The fixed cells are then suspended for two minutes at room temperature with 0.2N HC1. The suspended cells are denatured with a mixture of 70% formamide (ultrapure) and 30% 2X SC for 10 minutes at 70°C. The cells are immediately hybridized in hybridization buffer with six 5 nM Texas Red-labelled and (separately) six fluorescein-labelled 22-25 base probes specific for HSV1 and HSV2 for ten minutes at 60°C.
  • hybridization buffer with six 5 nM Texas Red-labelled and (separately) six fluorescein-labelled 22-25 base probes specific for HSV1 and HSV2 for ten minutes at 60°C.
  • oligonucleotides were labelled via the linker arm with isothiocyanate or sulfonyl chloride derivatives of either Texas Red or fluorescein by the method of PCT Publication No. WO 84/03285.

Abstract

Le procédé décrit permet de détecter des séquences d'acides nucléiques dans une cellule en suspension. Une technique pour l'hybridation directe de désoxyoligonucléotides marqués avec des séquences d'ADN ou d'ARN cellulaires contenues dans des cellules intactes en suspension ainsi que des techniques de séparation de cellules contenant l'ADN ou l'ARN hybridé des cellules qui ne contiennent pas d'ADN ou d'ARN hybridé sont décrites.
EP90905309A 1989-03-07 1990-03-05 Hybridation in situ en suspension pour la detection ou la separation de cellules Withdrawn EP0462221A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US31998289A 1989-03-07 1989-03-07
US319982 1989-03-07

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EP0462221A1 true EP0462221A1 (fr) 1991-12-27

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EP90905309A Withdrawn EP0462221A1 (fr) 1989-03-07 1990-03-05 Hybridation in situ en suspension pour la detection ou la separation de cellules

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EP (1) EP0462221A1 (fr)
JP (1) JPH04505556A (fr)
AU (1) AU646635B2 (fr)
CA (1) CA2011571A1 (fr)
WO (1) WO1990010715A1 (fr)

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CA2102428A1 (fr) * 1991-05-03 1992-11-12 Katherine W. Klinger Methode d'obtention de cellules informatives
BE1005174A3 (nl) * 1991-08-13 1993-05-11 Universitaire Instelling Antwe Werkwijze voor het opsporen van een virus in celmateriaal van een afschraapsel of lichaamsvocht.
US5501952A (en) * 1992-07-17 1996-03-26 Aprogenex, Inc. Analogues of reporter groups as background reducers in hybridization assays
AU4685593A (en) * 1992-07-17 1994-02-14 Aprogenex, Inc. Enriching and identyfying fetal cells in maternal blood for in situ hybridization
US5521061A (en) * 1992-07-17 1996-05-28 Aprogenex, Inc. Enhancement of probe signal in nucleic acid-mediated in-situ hybridization studies
AU4681393A (en) * 1992-07-17 1994-02-14 Aprogenex, Inc. Background-reducing compounds for probe-mediated in-situ fluorimetric assays
US5386024A (en) 1993-02-10 1995-01-31 Gen-Probe Incorporated Method to prepare nucleic acids from a biological sample using low pH and acid protease
CA2167804A1 (fr) * 1993-07-20 1995-02-02 Robert H. Singer Methode d¨hybridation d¨acide nucleique in vivo
EP0760008A1 (fr) 1994-05-19 1997-03-05 Dako A/S Sondes d'acide nucleique peptidique de detection de neisseria gonorrhoeae et de chlamydia trachomatis
US5582985A (en) * 1994-08-05 1996-12-10 The Regents Of The University Of California Detection of mycobacteria
EP0871770A1 (fr) * 1995-05-18 1998-10-21 Abbott Laboratories Sondes de peptides polymeres et leurs utilisations
US5888733A (en) * 1995-11-16 1999-03-30 Dako A/S In situ hybridization to detect specific nucleic acid sequences in eucaryotic samples
US6214563B1 (en) 1997-08-01 2001-04-10 Aurora Biosciences Corporation Photon reducing agents for reducing undesired light emission in assays
US6221612B1 (en) 1997-08-01 2001-04-24 Aurora Biosciences Corporation Photon reducing agents for use in fluorescence assays
US6200762B1 (en) 1997-08-01 2001-03-13 Aurora Biosciences Corporation Photon reducing agents and compositions for fluorescence assays
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US8953866B2 (en) 2004-03-16 2015-02-10 Amnis Corporation Method for imaging and differential analysis of cells
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Also Published As

Publication number Publication date
CA2011571A1 (fr) 1990-09-07
AU646635B2 (en) 1994-03-03
JPH04505556A (ja) 1992-10-01
WO1990010715A1 (fr) 1990-09-20
AU5289990A (en) 1990-10-09

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