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WO1993017128A1 - Procedes de detection de structure et de reagencements chromosomiques - Google Patents

Procedes de detection de structure et de reagencements chromosomiques Download PDF

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Publication number
WO1993017128A1
WO1993017128A1 PCT/US1993/001718 US9301718W WO9317128A1 WO 1993017128 A1 WO1993017128 A1 WO 1993017128A1 US 9301718 W US9301718 W US 9301718W WO 9317128 A1 WO9317128 A1 WO 9317128A1
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Prior art keywords
dna
chromosome
label
probe
signal
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PCT/US1993/001718
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English (en)
Inventor
Douglas J. Taron
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Amoco Corporation
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Priority to EP93906233A priority Critical patent/EP0612357A1/fr
Priority to JP5515084A priority patent/JPH06507318A/ja
Publication of WO1993017128A1 publication Critical patent/WO1993017128A1/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
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • This invention relates to methods for detecting a site characterized by a genetically significant rearrangement event in targeted chromosomal DNA sequences which may occur at any location in any chromosome.
  • This invention further relates to methods comprising steps of applying first and second labelled probes to a target nucleic acid at regions adjacent to said site, wherein the probes comprise DNA sequences which are complementary to the chromosomal DNA sequences of interest.
  • the labelled probe DNAs are then specifically associated with first and second interdependent signal producing moieties capable of interaction by the diffusion of a chemical substance to produce a detectable signal. With the addition of reagent , the first and second moieties are induced to produce a signal at the site of a genetically significant event, and the presence or absence of the signal is then optically detected.
  • This invention also relates to methods for revealing pre-existing fluorescent labels, both for revival of faded labels and confirmation of previous results.
  • Chromosome structure is intimately related to the manner and . mechanics of gene expression in normal cell function. Just as importantly, conservation of chromosome structure during cell division is necessary for transmission of genetic information from cell to cell, and from generation to generation. Often however, chromosome structure is changed and may forecast problems in gene function.
  • chromosome structure often coincide with, and may be the cause of many inborn genetic disorders and degenerative diseases, including certain cancers. Such alterations may take the form of additional or absent whole chromosomes, or additional or absent portions of chromosomes. Chromosomes may also be rearranged, as by a translocation, so that different chromosomal regions come to be linked to each other. A host of other genetic defects, including inversions, amplifications, and outright deletions, can occur alone or in combination with the above named defects.
  • chromosomal alterations are detectable as diseases. Alterations such as additional or absent chromosomes may lead to, for example, Down syndrome (extra chromosome 21 matter), Turner syndrome (deleted X chromosome in females) or Klinefelter syndrome (XXY chromosomes). Alterations involving parts of chromosomes can produce, for example, chronic myeiogenous leukemia (C L) and acute lymphocytic leukemia (ALL), both thought to occur in the presence of the so-called Philadelphia chromosome, which involves a translocation between chromosome 9 and chromosome 22.
  • C L chronic myeiogenous leukemia
  • ALL acute lymphocytic leukemia
  • Karyotype analysis is currently used in diagnosis of the aforementioned maladies.
  • a karyotype is essentially a tally of the number and characteristics of an individual's chromosomes.
  • Conventional karyotype analysis is done by staining and visualizing metaphase chromosomes and the characteristic patterns (called bands) produced. See, for example, ACT Cytgenetics Laboratory Manual, 2nd Edition, at page 222, Margaret J. Barch. Ed.. (1991 ) Raven Press Ltd., New York, New York.
  • the present invention provides methods for overcoming such limitations.
  • the present invention requires a simple light microscope for it's immediate application and, in addition, can readily be adapted to automation already available. The degree of training and judgement required of the analyst is much reduced as well.
  • FISH Fluorescence In-Situ Hybridization
  • the important disadvantages that remain are; (1), the inability to distinguish rearrangements involving smalt regions of chromosomal material, as in cytochemical banding; (2), in the case of fluorescence labelled probes for FISH, the requirement for expensive fluorescence optics on the microscope, and; (3) the inherent difficulty of using certain high-sensitivity stains without obfuscating chromosome morphology necessary for karyotypic analysis. Accordingly, there is a need to deal with such. shortcomings before the in-situ methodology can mature into a routine, but extremely valuable clinical tool.
  • the present invention has no such limitations, as it is an important and novel approach to the specific and precise labelling of such chromosomal rearrangements, and in addition requires a relatively simple phase optics equipped microscope.
  • Carr, EPO 0 246 864 discloses a method for using DNA partial hybrid probes to locate complementary target sequences of interest.to form "split probes" that can be linked together to make a detectable signal in the form of a double stranded DNA with high thermal stability, or other distinguishing physical character. Again, this method requires that precise information on the region of interest be in hand before application can occur.
  • Ullman, EPO 0 230 768 discloses methods of separating substances from a liquid medium, in which the presence of desired aggregates is determined by the formation of so-called complementary specific binding pairs, or "sbp's", which have been conjugated to selected enzymes.
  • the sbp's are detected by a signal producing system comprising the combination of enzymes linked to sbp's that interact to produce a measurable signal, or product.
  • No application of interacting enzyme pairs for use with DNA probes to chromosome structure is mentioned, although a DNA-DNA or DNA-RNA hybrid is mentioned as a possible sbp.
  • Gray, et.al., EPO Application No.90308718.7. discloses methods and compositions for chromosome specific staining using "direct" fluorescence labelled probes comprising high complexity DNA sequences from individual human chromosomes.
  • the specific applications call for the detection of chromosomal rearrangement by the microscopic detection of two different (color) fluorescence signals emanating from adjoining regions of FISH treated chromosomes, but the development of fluorescence does not depend on the presence of a rearrangement site.
  • Wiegant, et al, Nucleic Acids Res. 19, 3237 (1991), discloses the use of fluorescein-dUTP in a nick-translation format to produce fluorescein labelled human nucleic acid probes.
  • the probes are used for • in-situ hybridization of human metaphase chromosomes, and also serve as targets for cytoimmunologicai enhancement via anti-fluorescein antibodies carrying yet more fluorescein labels.
  • a more specific object of this invention is to provide a means of diagnosing chromosomal aberrations such as translocations wherein regions of different chromosomes become linked, sometimes causing or thought to cause disease
  • the objects of this invention can be attained by detecting a site characterized by a genetically significant rearrangement event in targeted chromosomal DNA sequences, which site may occur at any location in any chromosome, by applying the steps comprising:
  • step (b) contacting the labelled product of step (a) with first and second interdependent signal producing moieties, said first interdependent signal producing moiety (ISPM) capable of attaching specifically to said first label by immunological means, and said second interdependent signal producing moiety (ISPM) capable of attaching specifically to said second label by immunological means, wherein said first moiety and said second moiety are capable of interaction by the diffusion of a chemical substance to produce a detectable signal,
  • This invention provides methods, reagents and compounds for in- situ detection of a chromosomal translocation.
  • the reagents comprise unhybridized high or moderate complexity probe DNA sequences which
  • ⁇ are essentially complementary to most or all regions of the chromosome or chromosome region to be detected.
  • Complexity of probe DNA refers to the number of bases in sequences that are not repeated. Such probe DNA's are named whole chromosome paints (or WCP's tm Imagenetics-
  • whole chromosome paints refers to a probe or probe composition, such as a probe composition of this invention, which is adapted to contact or hybridize a target which comprises one predetermined (i.e., preselected) chromosome of a multi-chromosomal genome.
  • a predetermined (i.e., preselected) chromosome of a multi-chromosomal genome typically, one WCP of this invention is combined with a second
  • WCP so as to make possible the indirect staining and subsequent detection of one or more predetermined chromosomal regions.
  • the labelled probe DNAs useful in this invention comprise two essential moieties, namely a polynucleotide portion and a chemically combined label portion-
  • the polynucleotide portion of the probe DNAs can be in the form of plasmids. cosmids. phagemids, yeast artificial chromosomes (YACs) or other episomal DNA fo7ms, as well as DNA fragments of large or small size, that can adequately locate (i.e. hybridize) to specific chromosomal target sequences in sufficient quantity and juxtaposition to serve the purposes on this invention.
  • Preferred probe DNAs are whole chromosome paints, comprising high to moderate complexity DNA sequence fragments which are complementary to the chromosomal DNA sequences of interest-
  • the sources of the DNA sequence used in the invention include but are not limited to DNA isolated from specific chromosomes, or libraries of such DNA, prepared by methods well known to those with skill in the art.
  • the individual chromosomes from which DNA is isolated can be prepared by any of a number of standard methods, such as flow cytometry of microcell or somatic ceil hybrids, or by direct isolation from individual metaphase or interphase cells.
  • Another source of such DNAs are libraries of specific chromosomal DNA, prepared by standard methods and available from traditional sources known to those in the art, such as the American Type Culture Collection (ATCC) or other repositories of human or other cloned genetic material. While a large number of chromosome libraries are available from the ATCC, representative libraries are:
  • the ATCC deposits are available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland.
  • the invention contemplates that such DNA sequences may also be synthesized in - vitro by any of a number of enzymatic means known to those in the art. Also see an article entitled Human Chromosome-Specific DNA Libraries,
  • DNA used in the invention is isolated from these sources by methods which are well known to those skilled in the art.
  • This DNA is then reduced to a heterogeneous mixture of variably sized fragments by any of a number of physical, chemical or enzymatic treatments, including but not limited to sonication, limited DNase I digestion, limited mung bean nuclease digestion, and sheanng of DNA through a narrow-gauge needle.
  • the resulting mixture of DNA fragments are in a size range of 100-500 basepairs (bps) in length, although the preferred length of the average size of a fragment is about 300 bps.
  • the DNA fragments are first derivatized by any of a number of chemical means known to those in the art to provide the DNA fragments with moieties capable of covalently bonding with appropriate labels, preferably by transamination of the carbon 4 (C-4) atom amino group of the nucleotide base cytosine.
  • the derivatization results in the addition of a variety of reactive monoamme or diamine compounds at the C-4 position in this base, including but not limited to such compounds as hydrazine, alkylene diamines having 2 to 10 carbon atoms such as ethylenediamine, certain amino acids such as lysine or glutamine, and peptides, ether derivatives, or any of a number of other organic or inorganic linker molecules.
  • the DNA fragments have 5-25% of the cytosine residues contained therein transaminated.
  • the transaminated DNA sequences are covai ⁇ ntly linked to any of a number of labels comprising all compounds or entities which have a functional group capable of covalent bond formation with the transaminated DNA sequence, and are able to act as haptens in addition to other functionalities they may possess.
  • hapten is meant any chemical species that is able to be recognized and bound by an antibody, but which is not sufficient to illicit an immune response.
  • labels include but are not limited to, biotin (which may also interact with avidin and avidin-enzyme conjugates), phenyl and phenyl derivatives, caffeine and related compounds, fluoresciens, rhodamines and other fluorescent species, mercury or other metals, and any of a series of isoprenoids including carotenoids, sterols and steroids.
  • biotin which may also interact with avidin and avidin-enzyme conjugates
  • phenyl and phenyl derivatives caffeine and related compounds
  • fluoresciens rhodamines and other fluorescent species
  • mercury or other metals mercury or other metals
  • isoprenoids including carotenoids, sterols and steroids.
  • CMR- carboxytetramethylrhodamine
  • transaminated DNA sequences are reacted with an excess of functionalized label compounds and 60-80% of transaminated sites are labeled.
  • labels that are attached to previously hybridized DNA direct label probes clearly can also serve as the target of the ISPM (enzyme)-antibody conjugates in the dual enzyme system described above.
  • a direct label probe is one that is designed to stain or otherwise distinguish the target DNA without subsequent labelling steps, such as with fluorescent labels.
  • Many of the labels described in the prior art will operate well in such an application, since the only requirement would be that such labels are also haptens.
  • a peroxidase type of ISPM in combination with added hydrogen peroxide and chromogenic substrate can be used as a chromogenic system that targets fluorescent labels, specifically carboxytetramethylrhodamine (CTMR) and fluorescein and generally any of a series including rhodamiries, fluoresceins, umbelliferines, etc.
  • CTMR carboxytetramethylrhodamine
  • fluorescein specifically carboxytetramethylrhodamine
  • the aforementioned enzyme-antibody conjugate system operates on "faded" fluorescent labels, as well as those freshly prepared.
  • an anti- fluorescent label system allows recovery of the information for reanaiysis or confirmation from "old and faded" FISH treated metaphase spreads.
  • the presently described method also provides a secondary analytical tool for FISH treated metaphase spreads.
  • This invention relates to the use of a multiplicity of different chromosome-specific probes having distinct antigenically active labels. Such specified labelled probes are hybridized to chromosomes or chromosomal regions, such as those involved in translocations and rearrangements.
  • this invention relates to "in situ hybridization" of these chromosome specific probes to chromosomes from disrupted cells that have been prepared so as to leave the native chromosome structure essentially intact and to preserve the physical relationships between different chromosomes, different portions of the same chromosome or between chromosomes and other cellular structures.
  • m siiu hybridization refers to the contacting or hybridization of a probe to a target in which hybrids are produced between a probe and a target.
  • situ hybridization is inclusive of denaturation and of a hybrid or probe detection procedure which is practiced after iasiiu hybridization of a probe to a target.
  • a specimen can be adhered as a layer upon a slide surface.
  • Targets for this hybridization include but are not limited to chromosomes or regions of chromosomes in normal, diseased or malignant human or other animal or plant cells, either interphase or at any stage of meiosis or mitosis, and either extracted or derived from living or postmortem tissues, organs or fluids; germinal ceils including sperm and egg cells, seeds, pollen, or zygotes, embryos, chorionic or amniotic cells, or cells from any other germinating body; cells grown in vitro, from either long-term or short-term culture, and either normal, immortalized or transformed; inter- or i ⁇ traspecific hybrids of different types of cells or differentiation states of these cells; individual chromosomes or portions of chromosomes, or translocated, deleted or _, ⁇ O 128
  • chromosomes isolated by any of a number of means known to those with skill in the art, including libraries of such chromosomes cloned and propagated in prokaryotic or other cloning vectors, or amplified in vitro by means well known to those with skill; or any forensic material, including but not limited to semen, blood, hair or other samples.
  • the labeled DNA sequences Prior to hybridization, the labeled DNA sequences are preferably reacted with an excess of corresponding unlabeled DNA or reassociated fraction of unlabeled DNA for blocking non-specific hybridization.
  • This blocking DNA is used at a concentration of 1-10 micrograms per 10 microliters of total genomic DNA, with a preferred range depending on the hybridized chromosome.
  • the blocking DNA may be human placental DNA or Cot1 DNA (Cot . DNA supplied by Life Technologies, Gaithersburg, MD, Cat. # 5279SA). Briefly, Cot1 DNA is prepared by mechanically shearing total human genomic DNA to an average size of less than 400 base pairs.
  • This material is denatured and then rehybridized for a period sufficient to render a large fraction of the highly repeated DNA sequences double-stranded.
  • the mixture of double and single-stranded DNA species are treated with nuclease S1 , a nuclease that specifically degrades unhybridized single-stranded DNA to mono- and oligo-nucleotides. ' Undigested, double stranded Cot1 DNA is recovered from this mixture.
  • the present invention addresses problems in the detection and identification of chromosomal regions which can be involved in translocations and rearrangements.
  • the invention operates by producing signals resulting from the interaction of two interdependent signal producing moieties ("ISPM's").
  • the interdependent signal producing moieties can include catalysts, usually enzymes, and a plurality of substrates, and includes combinations of enzymes capable of interaction when the substrate of one enzyme is the product of the other enzyme.
  • the final product of such interaction is the detectable signal, usually a visible dye or light signal, or a reactive chemical species able to interact with additional added components.
  • the ISPM's are two interdependent (i.e. coupled) enzymes.
  • Combinations of enzymes that are of particular 5 interest include those which produce hydrogen peroxide and those which are able to use the hydrogen peroxide to oxidize a clear soluble substance to a detectable colored substance, such as a dye or other indicator.
  • peroxide producers include, but are not limited to, glucose oxidase, gaiactose oxidase, aldehyde oxidase, xanthine 0 oxidase, monoamine oxidase, dihydroorotate dehydrogenase, and L- and D-amino acid oxidases.
  • Examples of peroxide utilizers include horseradish peroxidase, microperoxidase, and cataiase.
  • Horseradish peroxidase is of particular interest because it can utilize a number of other compounds in addition to, or in conjunction with, 5 peroxide.
  • the enzyme alkaline phosphatase is able to convert 4-cJ ⁇ loro-napthyl-1 -phosphate to 4-chloro-napthol.
  • 4- chloro-napthol is converted to a an insoluble , dark purple dye, revealing the site of interest.
  • horseradish peroxidase is paired with glucose oxidase.
  • glucose oxidase coverts ⁇ -D- glucose to D-giucono- ⁇ )-lactone and hydrogen peroxide.
  • Horseradish peroxidase uses the hydrogen peroxide in conjunction with tetramethyibenzidine to produce a dark blue dye, and again reveals the
  • the aforementioned enzymes are conjugated to specific antibodies that correspond to chromosome specific probes.
  • a visible signal is produced that is detectable using
  • fluorophores as fluorophores, chromophores, chemiluminescent groups, odoriferous compounds 1 , and others which have properties facilitating detection.
  • Embodiments of this invention utilize the compounds dinitrophenyi (DNP), theophylline, carboxytetramethylrhodamine (CTMR), or fluorescein for labelling of high complexity DNA probes.
  • DNP dinitrophenyi
  • CMR carboxytetramethylrhodamine
  • fluorescein fluorescein for labelling of high complexity DNA probes.
  • antibodies target the said enzyme activities to the hybridized probes with anti-theophylline, anti- dinitrophenol, anti-CTMR, or anti-fluorescein immunoglobuli ⁇ s (IgG's).
  • Such reagent enzymes act either interdependently in the coupled system, or independently in the single system, to develop an easily visualized signal.
  • An important application of this invention is provision of a means to diagnose chromosomal aberrations such as translocations wherein regions of different chromosomes become linked, sometimes causing or thought to cause disease.
  • chromosomal aberrations such as translocations wherein regions of different chromosomes become linked, sometimes causing or thought to cause disease.
  • diseases are chronic myeiogenous leukemia (CML) and acute lymphocytic leukemia (ALL), both thought to occur in the presence of the so-called Philadelphia chromosome, which involves a translocation between chromosome 9 and chromosome 22.
  • CML chronic myeiogenous leukemia
  • ALL acute lymphocytic leukemia
  • the breakpoints associated with this and other similar translocations are known to occur over a range of up to 150,000 bases. This fact obviates the use of so-called specific DNA probes for broad utility, since the fine structure of the breakpoint region would be required to target such discrete probes on their own.
  • samples of chromosomes comprising the target DNA are prepared from cells of interest (for example, CML or ALL) and placed on a solid support such as a slide using well known in-situ fixation methods.
  • Two of the above described labelled DNA probes are then hybridized to the fixed target DNA in the form of chromosomes, and sequentially treated with immunological reagents carrying interdependent (coupled) enzyme activities and reagents to catalyze a signal forming reaction, only in those chromosomal regions where a translocation has placed the enzymes - . .
  • a chromosome having a translocation appears to have a colored or stained segment attached to an unstained remainder segment, and the complementary pattern also appears on a corresponding chromosome.
  • a WCP probe for chromosome 1 was labelled with dinitrophenyl and hybridized to a normal lymphocyte metaphase spread. After hybridization, anti-dinitrophenyl goat IgG was applied to the hybridized labelled probe. Anti-goat IgG conjugated to horseradish peroxidase was 1 5 then reacted to the previously bound goat anti-dinitrophenyl antibody. The entire preparation was then perfused with a solution containing alkaline phosphatase. 4-chloro-napthol, and hydrogen peroxide. The reaction produced intense black staining over chromosome 1 , and very little background staining of other chromosomes.
  • a translocation between chromosome 1 and chromosome 4 was detected using the present invention.
  • the cell line sup B13 which contains the chromosome 1-4 translocation was the source of chromosomes for the preparation of metaphase spreads.
  • Two different whole chromosome paint (WCP) was used to detect a translocation between chromosome 1 and chromosome 4 .
  • WCP for chromosome 1 was labelled with theophyliine. and WCP for chromosome 4 was labeled with dinitrophenyl (DNP).
  • DNP dinitrophenyl
  • HRP horseradish peroxidase
  • GOX glucose oxidase
  • Elements of the present invention also provide a methodology that will amplify, retrieve or otherwise recover normally faded fluorescent in- situ hybridized (“FISH") labelled metaphase slides by restaining with the coupled enzyme chromogenic system of this invention.
  • FISH fluorescent in- situ hybridized
  • Such an application utilizes, for example, the fluorescent labels of previously hybridized probes as haptens for the attachment by immunological means of one or more of the ISPM's heretofore described.
  • the method thus provided comprises the steps of contacting flourescent labels bound to previously hybridized probes with a signal producing moiety, wherein said moiety is directed to flourescent labelled chromosomes by immunological means comprising antigen/antibody or antibody/anti- antibody pairs, and reacting reagent comprising a first and second substance with said moeity. thereby converting a colorless soluble substrate to an insoluble detectable signal, said signal being in the range of visible light and detectable by optical means.
  • Human chromosome-specific DNA probes were obtained as recombi ⁇ a ⁇ t phage libraries from Lawrence Livermore National Laboratories (LLNL) constructed as described in Van Diila, M.A. ⁇ t al. (Biotechnology 4: 537-552, 1986). These libraries were amplified by growth on an E. coli host strain. The amplified phage were purified, their DNA was extracted, and this DNA was digested with the restriction enzyme Hind III. Insert DNA was purified away from the lambda vector DNA and cloned into the Hind III site of the plasmid vector pBS (Strategene, La Jolla, CA). The resulting piasmids were transformed into an E. coli strain, DH5 ⁇ (Bethesda Research Libraries, Gaithersburg, Maryland). The plasmid libraries used in this example are ATCC #'s 57738,
  • the libraries are stored as 1 ml aliquots of frozen cells. These vials have been used as the primary source for the production of seed stocks for fermentation.
  • Bacteria were grown by fermentation.
  • the seed stock obtained from ATCC was cultured at 37°C for 24 hr. on 1.6% agar plates containing ampicilli ⁇ (200 microgram/mi) and YT broth, which contains 8 grams per liter (g/l) of Bacto Tryptone (Difco), 5 g l of Bacto Yeast Extract (Difco), 15 g/l of Bacto Agar (Difco), and 5 g/l of sodium chloride.
  • the cultured cells were harvested with 4 ml containing 16 g/l of Bacto Tryptone (Difco), 10 g/l of Bacto Yeast Extract (Difco) and 5 g/l of sodium chloride, and 4 ml of 20% glycerol was added to each harvest.
  • the E. coli cell culture was quickly frozen in 0.5 ml aliquots by submerging the vials in liquid nitrogen and stored at -80°C until use.
  • the fermenter inoculum was prepared in 350 ml by cutturing the seed culture in a Casamino Acid medium which contains 13.2 g/l Na 2 HPO4-7H 2 0, 3.0 g/1 KH 2 PO 4 . 0.05 g/l NaCI, 1.0 g/l NH 4 CI, 10.0 g/l Casamino Acids (Difco); 0.03 g/1 MgSO 4 . 0.004 g l CaCI 2 -2H 2 0, 3.0 g/l glucose, 0.025 g/l Thiamin ⁇ -HCI.
  • Bacterial cells were harvested employing a membrane cell- concentrator and a high speed centrifuge immediately after completion of the fermentation.
  • the fermented cell broth was concentrated from 5 liter to approximately 800 ml employing a 0.45 micron (_m) membrane filter (2 square feet).
  • the cell concentrate was then centrifuged at 7,000 x g for 10 minutes in a refrigerated centrifuge. The bacterial cell pellets are recovered after discarding the supernatant. 8
  • Plasmid DNA was extracted from bacterial cell pellets.
  • the cells were thoroughly resuspended in 3 times the cell pellet mass (M) (in milliliters) of a solution containing 50mM glucose (fitter sterilized), 10 mM NaEDTA (pH 7.5-8.0), and 25mM Tris-HCI (pH 8.0).
  • M cell pellet mass
  • the cells were lysed with vigorous swirling after the addition of 6xM (in milliliters) in a solution containing 0.2 M NaOH, and 1% (w v) sodium dodecylsulfate (SDS).
  • nucleic acid was precipitated from the supernatant with one volume of ethanol followed by centrifugation for 10 minutes at 7000 x g, and the nucleic acid pellets were resuspended in a total of 0.54xf "in milliliters). The nucleic acid was then extracted with 1/2 volu of neutralized phenol and 1/2 volume of chloroform and precipitated with two volumes of ethanol. The nucleic acid was resuspended in 0.3xM (in milliliters) of a solution of 50 mM Tris HCI (pH 7.0) and 100 mM sodium acetate.
  • DNA v .ia resuspended in 0.415xM (in milliliters) of water, and 0.05xM milliliters of 5 M NaCI and 0.155xM milliliters of 50% (w/v) polyethyleneglycol (PEG) (molecular weight 6000-8000) were added, incubated on ice water for one hour and precipitated by centrifugation for 15 minutes at 7,000 x g.
  • the DNA was resuspended in 0.04xM milliliters of water and 1/10 volume of 3M sodium acetate and extracted with 1/2 volume of neutralized phenol and 1/2 volume of chloroform and precipitated with two volumes of ethanol.
  • the purified DNA was resuspended in 0.0476xM milliliters of deionized H 2 0. The DNA concentration was determined by fluorometry. 8
  • the purified DNA was disrupted into small fragments of approximately 300 base pairs by sonication using a Branson Sonifier 450 (Danbury, Connecticut). This size of fragments has been empirically determined to be the optimum for DNA probes used for in situ hybridization.
  • Four milligrams of the purified plasmid DNA prepared above was resuspended in 2 mis of water and immersed in a dry ice/ethanol bath to prevent boiling during sonication. The microtip of the sonication device was- immersed in this solution until the tip was 2-5mm from the bottom of the tube.
  • Sonication was carried out at an output power of 25-30 watts, discontinuously, with an 80% duty cyle (on 80% of time, off 20% of time), for a period of 5 minutes.
  • the DNA was precipitated by the addition of 0.2 ml of 3 M sodium acetate (pH 5.5) and 4 ml of ethanol. The precipitate was recovered by centrifugation for 5 minutes at 8,000 x g and vacuum dried.
  • DNA obtained by the method of Example 1 was transaminated by the addition of ethylenediamine to the C4 carbon atom of the base cytosine. This reaction is catalyzed by sodium bisulfite. To prepare the bisulfite buffer, 1.7 ml of fuming HCI was slowly added to 1 ml deionized
  • the transamination reaction was initiated by the addition of 0.3 mi of this DNA solution to 2.7 ml of bisulfite buffer, and the reaction was incubated at 37°C for 2 days.
  • the DNA solution was desalted by routine dialysis against 5-10 miliimolar sodium borate (pH 8.0). After dialysis, 0.3 ml of 3 M sodium acetate (pH 5.5) was added to the diaiysate.
  • the aminated DNA was precipitated with 2.5 volumes of ethanol and recovered after centrifugation at 8,000 x g for 10 minutes. The pellets were vacuum dried and rehydrated at a concentration of 3 mg/ml DNA. This solution was stored at -80°C until use.
  • a solution of 20 mM ⁇ -amino- ⁇ -caproic acid was prepared by adding 2.62 g of this compound to 20 ml water containing 40 mmol sodium bicarbonate. This solution was mixed with 20 ml of a 20 mM solution of Sanger's reagent (2,4 dinitro-fiuorobenzene) and allowed to stand at room temperature for 1 hour. The mixture was then gently heated, which caused the solution to turn yellow and a small amount of the dissolved sodium bicarbonate to precipitate. This precipitate was re- dissolved by the addition of a sufficient quantity of concentrated HCI and then left at 4°C to induce crystallization. The crystals were collected in vacua and washed with water to yield 4.2 g of yellow crystalline ⁇ - dinitrophenylamino-t7-caproic acid (DNP-NCA).
  • DNP-NCA yellow crystalline ⁇ - dinitrophenylamino-t7-caproic acid
  • DNP-NCA was activated by esterification to 3-sulfo-N- hydroxysuccinimide as follows. 0.594 g of DNP-NCA, 0.468 g dicyclohexylcarbodiimide and 0.434 g of 3-sulfo-N-hydroxysuccinimide were vigorously stirred in 7 ml dimethylformamide at room temperature overnight. This reaction was determined to have gone > 90% to completion by thin layer chromato ⁇ raphy. The mixture was cooled to 0°C ' and stirred for an additional hour. The mixture was then filtered and the yellow solution evaporated to a thick yellow oil which did not crystallize.
  • This oil was stirred with 50 ml ethanol to yield 0.996 g of a fine yellow powder which was collected by filtration and washed with ethanol.
  • This compound is 6-N-(2.4 .1 ⁇ nitrophenylamino)caproic acid-O-(N- hydroxysuc ⁇ nimid ⁇ )-3-sulfo ⁇ .at ⁇ (sodium salt) and will be referred to for the purposes of this invention as S-NHS-DNP.
  • Example 4 DNA Labeling: DNP-Deriviatived ⁇ -amino-n-Caproic Acid
  • Chromosome-specific DNA of average length of about 300 bp prepared by the method of Example 1 was derivatized by bisulfite catalyzed transamination with ethylenediamine as described in Example 2.
  • a solution of aminated DNA (100 ⁇ g total DNA) in a plastic 1.5 ml centrifuge tube was evaporated under reduced pressure.
  • 0.5 ml of 0.2 M 3-[N-morpholino] propane sulfonic acid (MOPS) buffer and then 100 8
  • microliters of S-NHS-DNP (30mg/ml N,N-dimethyiformamide) was added to the residue and the mixture was incubated overnight at 25°C.
  • DNP- Labeled DNA was precipitated by the addition of 60 ⁇ l of 3 M sodium acetate (pH 5.5) followed by 1.5 ml of ice cold ethanol and the mixture was incubated for at least 2 hours at -20°C. The solution was subjected to centrifugation for 10 minutes at 10,000 x g. The DNA pellet was washed twice with 0.6 mi of ice cold ethanol and then dissolved in 100 ⁇ l of sterile water.
  • This solid was recrystailized from a boiling 1 :1 propanol/water mixture and dried over calcium sulfate to provide a colorless solid.
  • a solution of 0.488 gram of dicyciohexyl-carbodiimide (DCC) in 2 miililiter of anhydrous DMF was added to the above solution.
  • the precipitated material was collected by filtration and dried over anhydrous calcium sulfate to provide 0.450 gram of theophylline-8-N-(5- hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl)) ester, (NHS-theophylline).
  • Chromosome-specific DNA probes to human chromosome 4 of average length of about 300 bp obtained by the procedure- of Example 1 were derivatized with the bisulfite catalyzed transamination with ethylenediamine as described in Example 2. Approximately 5% of the bases were aminated. A solution of aminated DNA (100 micrograms total DNA) in a plastic 1.5 miililiter centnfuge tube was evaporated under reduced pressure. 0.5.
  • Chromosome-specific DNA probes to human chromosomes 1 and 4 of average length of about 300 bp obtained by the procedure of Example 1 were derivat ⁇ zed by the bisulfite catalyzed transamination with ethylenediamine as described in Example 2. Approximately 5% of the bases were aminated. A solution of aminated DNA (50 micrograms total DNA) in a plastic 1.5 miililiter centrifuge tube was evaporated under reduced pressure. To this solution was added 377 microliter of 0.2 Molar 3-[N-morpholinoj propane sulfonic acid (MOPS) pH 7.4 buffer.
  • MOPS N-morpholinoj propane sulfonic acid
  • Transaminated DNA probes obtained by the method of Example 2 were conjugated with 5-(and-6)-carboxyfiuorescein, succinimidyl ester (CFI). Fifty micrograms of transaminated DNA were dried and then resuspended in 377 microliters of 200 mM MOPS, pH 7.4. Twenty-two and eight tenths microliters of 50 mM solution of 5-(and-6)- carboxyfluorescein, succinimidyl ester, (CFI) in N.N-dimethylformamide (a 150-fold molar excess) was added to the transaminated DNA. This reaction proceeded with stirring in darkness at room temperature overnight (approximately 18 hours).
  • the excess fluorophore was separated from the labeled DNA first by an ethanol precipitation.
  • the precipitated material was resuspended in water and passed over a Sephadex G-25 column that was 28 cm high with an internal diameter of 1 cm.
  • the desired fraction (the column void volume) was eluted in water and dried to reduce the total volume.
  • a second ethanol precipitation of the labeled DNA completed the purification. An absorbance spectrum showed that 1.6% of the bases were labeled.
  • SupB13 cells (CML line with multiple translocations case #10535) were made available by Michelle LeBeau, University of Chicago. Cells were reseeded into 25 cubic-centimeter flasks containing 80% RPMI 1640 media (Gibco catalog #320-1875), 20% fetal calf serum, 100 units Penicillin/Streptomycin, and 10 millimolar HEPES buffer. Cell growth was monitored by counting and cells were refed every 3 or 4 days. When cells had achieved optimal concentration, 0.2 ml 10 micrograms/milliiiter Colicimed (Gibco catalog#890-l 145-1) was added to each flask, and then held for 1 hour at 37C in a 5% CO2 incubator.
  • Cells were harvested by centrifugation and resuspended in 5 milliliters 75 millimolar KCI, then held for 10 minutes at 37C. Cells were again harvested by centrifugation, and all but 0.2 miililiter of the KCL supernatant was removed. The ceils were dispersed, then fixed slowly in 10 milliliters 3/1 methanol/acetic acid, and held on ice for 15 minutes. Cells were harvested by centrifugation and placed in 5 milliliters fresh methanol/acetic acid, and held another 15 minutes on ice. Cells were harvested, resuspended in methanol acetic acid, and placed dropwise onto precieaned glass slides and dried.
  • the coupled assay was tested by in situ hybridization using the standard technique.
  • the target DNA was present in the sup B13 metaphases on a glass microscope slide.
  • the slide was examined to locate areas containing nuclei and metaphase spreads. Outlines of the hybridization target area, and identification marks were made on the reverse side of the slide with a diamond scribe.
  • the target DNA on the slide was denatured by immersion for 2-10 minutes in a solution of 70% formamid ⁇ /0.3 M NaCI/30 millimolar sodium citrate, pH 7.6-7.8 at 70° C. Following denaturation, the target DNA was placed in a 70% ethanol/water bath and agitated to remove the formamide solution. The wash step was repeated by passing the slide through 70%, 85% and 8
  • the hybridization mixture consisted of 50% formamide/0.3 molar NaCI/30 millimolar sodium citrate, pH 7.0. Human piacental DNA (2.25 microgram/10 microliter) was used as the blocking DNA.
  • the theophylline labelled WCP 1 and the DNP labelled WCP 4 were each added to a concentration of 10 nanogram/microfiter.
  • the total hybridization volume was 10 microliter.
  • the hybridization mixture was denatured for 5 minutes at 70° C, then added to the slide over the area which contained the target DNA. A coverslip was placed on the hybridization mixture, and the edges were sealed with rubber cement. The slide was placed in a humidified chamber and hybridization proceeded overnight at 37° C.
  • the coverslip was removed and the slide was washed three times (5 minutes each) in 50% formamide/0.3 M NaCI/3 millimolar sodium citrate, pH 7.0 at 45° C. The slide was then washed 5 minutes in 0.3 M NaCI/3 millimolar sodium citrate and 5 minutes in 0.1 M sodium phosphate/0.1% NP40 (PN Buffer) each at 45° C. The slide was washed twice in PN buffer at room temperature, 2 minutes each wash. The slide was incubated for 20 minutes in anti-dinitrophenol (rabbit) diluted 1:250 in PNM buffer (PN buffer with 5% nonfat dry milk). The slide was washed 3 times in PN buffer at ambient temperature for 2 minutes each time.
  • PN Buffer sodium phosphate/0.1% NP40
  • the slide was then incubated for 20 minutes in glucose oxidase-ant ⁇ rabbit conjugate, and again washed 3 times in PN buffer.
  • the slide was finally incubated for 20 minutes in horseradish peroxidase-anti theophylline conjugate and washed a final 3 times in PN buffer, with fresh buffer used for each washing step.
  • the TMB treated metaphase spread was counterstained for 5 minutes in freshly prepared, filtered Giemsa.
  • the slide was rinsed under a gentle stream of distilled water.
  • the slide was then dried with an air or nitrogen jet.
  • the staine ⁇ metaphase spread was then examined in a microscope.
  • At least two chromosomes were labelled in all metaphases observed. In each case, only a portion of the chromosome was labeled, the labelled portion corresponding to that part of each chromosome derived from chromosome 1 . In some metaphases, a third chromosome was detectably labelled. In each case, this third chromosome was the normal copy of chromosome 1 .

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Abstract

L'invention se rapporte à des procédés et des réactifs utilisés pour la détection in situ d'une structure chromosomique ou d'une région chromosomique présentant des réagencements. Ces réactifs comprennent une multiplicité de séquences sondes d'ADN marquées qui sont complémentaires à différentes parties du chromosome ou de la région à détecter, ainsi que les anticorps spécifiques aux éléments de marquage, conjugués à des fractions interdépendantes produisant un signal. Des paires de sondes sélectionnées sont mises en contact dans des conditions d'hybridation, avec le chromosome ou la région chromosomique à étudier. Lesdits anticorps spécifiques aux éléments de marquage sont ultérieurement fixés à ces derniers, produisant ainsi la combinaison des réactions chimiques subies par lesdites fractions lors de l'addition de substrats. Un signal est ainsi produit au niveau de la zone chromosomique à étudier, et peut être détecté par des moyens optiques.
PCT/US1993/001718 1992-02-28 1993-02-25 Procedes de detection de structure et de reagencements chromosomiques WO1993017128A1 (fr)

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JP5515084A JPH06507318A (ja) 1992-02-28 1993-02-25 染色体構造および転位の検出方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018867A1 (fr) * 1994-01-10 1995-07-13 Celsis International Plc Dosage par hybridation et reactifs
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
US7034144B2 (en) 1997-05-13 2006-04-25 Erasmus Universiteit Rotterdam Molecular detection of chromosome aberrations
US7105294B2 (en) 1998-05-04 2006-09-12 Dako Denmark A/S Method and probes for the detection of chromosome aberrations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986003227A1 (fr) * 1984-11-23 1986-06-05 Dgi, Inc. Analyse des sequences d'acide nucleique, notamment des lesions genetiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986003227A1 (fr) * 1984-11-23 1986-06-05 Dgi, Inc. Analyse des sequences d'acide nucleique, notamment des lesions genetiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CYTOGENETICS AND CELL GENETICS vol. 54, 21 December 1990, BASEL, CH pages 108 - 111 E.P.J. ARNOLDUS ET AL. *
MOLECULAR BIOLOGY AND MEDICINE vol. 7, no. 5, October 1990, EXETER, UK pages 423 - 435 S.S.M. HABEEBU ET AL. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018867A1 (fr) * 1994-01-10 1995-07-13 Celsis International Plc Dosage par hybridation et reactifs
AU680988B2 (en) * 1994-01-10 1997-08-14 Celsis International Plc Hydridisation assay and reagents
US7034144B2 (en) 1997-05-13 2006-04-25 Erasmus Universiteit Rotterdam Molecular detection of chromosome aberrations
US7105294B2 (en) 1998-05-04 2006-09-12 Dako Denmark A/S Method and probes for the detection of chromosome aberrations
US7368245B2 (en) 1998-05-04 2008-05-06 Dako Denmark A/S Method and probes for the detection of chromosome aberrations
US7642057B2 (en) 1998-05-04 2010-01-05 Dako Denmark A/S Method and probes for the detection of chromosome aberrations
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

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CA2107880A1 (fr) 1993-08-29
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JPH06507318A (ja) 1994-08-25

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