WO1998041651A1 - Procede de preparation de chromatine - Google Patents

Procede de preparation de chromatine Download PDF

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Publication number
WO1998041651A1
WO1998041651A1 PCT/CA1998/000251 CA9800251W WO9841651A1 WO 1998041651 A1 WO1998041651 A1 WO 1998041651A1 CA 9800251 W CA9800251 W CA 9800251W WO 9841651 A1 WO9841651 A1 WO 9841651A1
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chromatin
cells
dna
spreads
fragmented
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PCT/CA1998/000251
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English (en)
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Thomas D. Yager
David W. Chan
Deborah L. Brooker
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Hsc Research & Development Limited Partnership
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • This invention describes a novel method for preparing spreads of eukaryotic chromatin, for a plurality of uses.
  • Free-chromatin methods for example that described by Heng & Tsui (USP # 5,470,709), have a very low yield. Approximately only 3-5% of cells lyse in such a way that useful fibres are produced. In contrast, with the present invention, useful spreads are produced from >50% of nuclei. This represents a 10 to 20-fold improvement in yield.
  • the prior art methods are highly specialized for the production only of spreads for FISH mapping. They do not, for example, allow the production of chromatin fibres or condensed chromatin bodies which can also be used in electrophoretic studies or in studies of the solid-liquid crystal transition in chromatin.
  • the present invention is general-purpose, and allows chromatin to be prepared for a multiplicity of uses.
  • Cell-free systems have been devised for the study of apoptosis (programmed cell death). Many such systems employ the fragmentation of cell nuclei as a specific endpoint that can be monitored by means of (1) cytological examination under the epifluorescence microscope, and (2) electrophoresis in agarose or polyacrylamide gels. (Lazebnik, Y.A. et al., 1993; Newmeyer, D.D. et al., 1994; Gromova, I.I. et al., 1995.)
  • the prior art does not describe methods to utilize the apoptosis phenotype as a diagnostic tool, to detect functional differences between wild-type cells, chemically- treated cells, and genetically mutant cells.
  • the present invention employs chromatin spreads, as substrates to reveal the normal or aberrant operation of the apopotic machinery of the cell. This could reveal the existence of upstream defects in the control pathway for apoptosis, thus forming the basis for a diagnostic assay.
  • the present invention utilizes the apopotic machinery of the cell, to enzymatically introduce precise cuts in chromatin and DNA fibres which are spread across the microdissection surface. This does not require application of physical force.
  • the prior art methods have not allowed the simultaneous isolation and examination of a range or variety of different DNA-containing objects.
  • the present invention allows all of the following objects to be purified, for use in subsequent electrophoretic and other transport studies: intact cell nuclei, individual prometaphase or metaphase chromosomes, individual long chromatin fibres, individual chromatin beads of a broad range of sizes.
  • the prior art methods involve either the tracking of a very large population (e.g., millions) of molecules so as to obtain statistically average behaviour; or the laborious tracking of individual molecules.
  • the current invention proposes a method to allow the simultaneous tracking of a plurality or multiplicity of similar objects spanning a range of sizes.
  • the present invention provides a method for generating a plurality of highly-condensed, micron- to sub-micron-sized chromatin particles, which can be induced to undergo a solid-to-liquid crystal transition.
  • This apparently reflects a new, previously undescribed phenomenon, which could have a practical utility, as a route to generating liquid crystalline "particles" for a variety of practical uses, e.g. as sensor elements.
  • the present invention relates to a novel method for preparing chromatin. Accordingly, the present invention provides a method for preparing chromatin from cells comprising (a) lysing cells in an aqueous solution; and (b) lysing the nuclei of the cells between two surfaces which are separated by an aqueous layer; and (c) allowing transverse fluid flow to occur between the surfaces, to release the chromatin which attaches to one or both surfaces.
  • the nuclei are preferably lysed in an aqueous solution containing a low to moderate salt concentration.
  • the temperature of the reaction is preferably about 2-10°C, although a broader temperature range may be tolerated if an exogenous agent is added to help lyse the nuclei.
  • At least one of the surfaces is preferably coated with a surface layer which has affinity for binding chromatin and DNA, for example poly(L-lysine) (Williams, 1977; Meng et al., 1995).
  • the composition at a surface layer is such that it can be made to form chemical crosslinks with the chromatin or DNA; for example, if the surface layer is poly(L-lysine), then it may be crosslinked to the chromatin or DNA by means of an aldehydic reagent such as formaldehyde (Solomon & Varshavsky, 1985; Kuykendall & Bogdanffy, 1992, 1994). It may also be crosslinked by means of UV irradiation (DeRisi, J.L. et al., 1997).
  • the chromatin prepared according to the present invention is advantageously obtained in a relatively intact state.
  • the two surfaces are optically clear, allowing a direct visualization of the spread chromatin by means of optical or epiflourescence microscopy.
  • the chromatin fibers may be continued to be incubated in order to allow for spontaneous cleavage of the DNA within the fibers, by means of residual apopotic machinery from the lysed cells, to produce fragmented chromatin or DNA-containing beads.
  • an agent that cleaves chromatin may be added to the aqueous solution, to produce this fragmentation.
  • An example of such an agent would be a deoxyribonuclease enzyme.
  • the present invention also provides a method for preparing fragmented chromatin comprising (a) lysing cells in an aqueous solution; (b) lysing the nuclei of the cells between two surfaces which are separated by an aqueous layer; (c) allowing transverse fluid flow to occur between the surfaces, to release the chromatin which attaches to one or both surfaces; and (d) incubating the chromatin fibers for a period of time sufficient for the cleavage of the DNA within the fibers.
  • a spontaneous fragmentation of the chromatin fibers can be produced through the activation of endogenous nucleases, which are components of the intact apopotic machinery of the lysed cells. These nucleases operate on the spread chromatin to convert it into an ordered one or two dimensional array of chromatin fragments or DNA-containing beads.
  • the chromatin fibers are preferably incubated at 2-10°C for at least 24 hours, and preferably for 24 to 72 hours.
  • the spontaneous cleavage can be blocked by several methods including: a transient pulse of heat and phosphatase; by EDTA or EGTA; or by adding one or more caspase inhibitors.
  • fragmentation can be induced by adding an exogenous agent that cleaves the DNA such as a deoxyribonuclease enzyme.
  • the chromatin beads can be moved through the aqueous medium between the two surfaces and thus be separated from one another, for example by means of electrophoresis. Further, the beads can be moved into pre-formed wells within the solid surfaces, and can then be used as substrates for PCR.
  • the chromatin and chromatin fragments or beads prepared according to the method of the present invention can be used in a variety of techniques including, but not limited to, (1) as a substrate for physical genome mapping, by means of sequence-specific hybridization of nucleic acid probes; (2) as a tool to investigate the natural organization of chromatin within cells; (3) to investigate changes in the natural organization of chromatin which result from the actions of drugs or other bioactive compounds on the cells; (4) as a tool to investigate normal or aberrant apoptosis in cells; (5) as an early-stage test within a hierarchical screening procedure, for the rapid detection of functional aberrations in the biochemical pathway which controls apoptosis; (6) to evaluate weak points in DNA, such as weak points associated with fragile-chromosomes syndromes such as the Fragile X syndrome; (7) for obtaining ordered chromosomal fragments, or for the isolation of chromosomal fragments such as those which specifically hybridize with a probe, or between pairs of adjacent probes; (8) as a substrate
  • the fragmented chromatin or ordered arrays of DNA containing beads produced according to the above method can be induced to detach from an underlying substrate or matrix. Once detached, the beads diffuse freely in solution. The diffusion can be tracked by means of an X-Y projection and translational diffusion co-efficients can be calculated.
  • the beads when raised in temperature from about 2-10°C to about 15-37°C exhibit a transition from solid to liquid crystalline phase. The transition from solid to liquid crystalline phase has not been previously described for chromatin.
  • the present invention provides a method for preparing liquid- crystalline chromatin comprising (a) lysing cells in an aqueous solution; (b) lysing the nuclei of the cells between two surfaces which are separated by an aqueous layer; (c) allowing transverse fluid flow to occur, to release the chromatin which attaches to one or both surfaces; (d) optionally perfusing in an agent which cleaves the chromatin to produce fragments; (e) incubating the chromatin for a period of time sufficient for the cleavage of the chromatin to produce fragments; and (f) heating the chromatin to a temperature sufficient to allow a solid to liquid crystal transition to occur in the chromatin fragments.
  • the temperature is raised from 2-10°C to at least 15°C, more preferably the temperature is raised to a temperature between 15°C to 37°C.
  • the liquid-crystalline chromatin particles comprise a unique system to allow the observation of solid - liquid crystal transitions within chromatin.
  • the liquid-crystalline chromatin particles might also find use in practical applications, for example as component parts within chemical sensor arrays. (Livingston GK, 1980; Zhu C & Hieftje GM, 1990; Tan, W. et al., 1992; Bronk KS & Walt DR, 1994; Healey, B.G. et al., 1995; Healey BG & Walt DR, 1997.)
  • the present invention also provides a method for preparing individual fibres of chromatin, and also webs and bundles of chromatin fibres, which display highly intricate, quasi-periodic or periodic substructures. It may be possible to use these objects as intermediate reagents in construction schemes in which DNA or chromatin templates are used to pattern the building of supramolecular objects or assemblages. For example, a "molecular addressing" scheme could be envisioned, in which the spatial position of an element in a supramolecular assembly is directed by sequence-specific hybridization to a known location in a genomic DNA matrix.
  • Figure 1A is a photomicrograph showing webs and bundles of fine fibers from interphase nuclear spreads from zebrafish embryos.
  • Figure IB is a set of digitization plots which reveal periodicities in the arrangement of fibers within webs and bundles from Figure 1A.
  • Figure 2A is a photomicrograph showing finely dispersed fibrous sheets from interphase nuclear spreads from zebrafish embryos.
  • Figure 2B is a topographic plot of digitized film negatives showing holes or defects in the fibrous sheets from Figure 2A.
  • Figure 3 is a photomicrograph showing the presence of DNA and histone HI in the nuclear spreads (left and right panels, respectively).
  • Figure 4 is a photomicrograph showing the presence of DNA in the nuclear spreads, revealed by means of TdT-mediated biotin-dCTP incorporation.
  • Figure 5 is a photomicrograph showing the fragmentation of nuclear spreads upon prolonged incubation at 4°C
  • Figure 6A-F is a set of graphs demonstrating the physical properties of beads produced by spontaneous fragmentation of nuclear spreads.
  • Figure 7 is a set of digitization plots which show measured periodicities of fragmentation of nuclear spreads.
  • Figure 8A is a photomicrograph showing the fragmentation of nuclear spreads during campothecin-induced apoptosis.
  • Figure 8B is a set of digitization plots which show residual periodicities in the nuclear spreads of Figure 8A.
  • Figure 9 is a photomicrograph showing thick, web-like structures that are produced by treatment of nuclear spreads with caspase-2, 3, 7 inhibitor Ac-DEVD-CHO.
  • Figure 10 is a photomicrograph which shows marginated nuclei that are produced by the treatment of nuclear spreads with caspase-2, 3, 7 inhibitor Ac-DEVD- CHO.
  • Figure 11 is a photomicrograph which shows a striated matrix that underlies the beads, produced by treatment of the nuclear spreads with the caspase-2, 3, 7 inhibitor Ac-DEVD-CHO.
  • Figure 12A-C are photomicrographs which show a liquid-crystalline behaviour of chromatin beads, which is induced by raising the temperature from an initial value of 2-10°C, to a final value of 15-37°C
  • Figure 13 is a photomicrograph of interphase nuclei derived from embryos incubated in Br-dU; the nuclei were stained with anti(Br-dU), followed by TRITC- conjugated anti(IgG).
  • the present invention relates to a novel method for preparing chromatin.
  • the present invention provides a method for preparing chromatin from cells comprising (a) lysing cells in an aqueous solution; and (b) lysing the nuclei of the cells between two surfaces which are separated by an aqueous layer; (c) allowing transverse fluid flow to occur between the surfaces, to release the chromatin which attaches to one or both surfaces.
  • the cells may be derived from embryos of a multicellular eukaryotic organism.
  • the cells are in an unfixed live state at the time of lysis.
  • the cells are preferably lysed in an aqueous solution containing a low to moderate salt concentration.
  • the temperature is preferably less than 24°C, more preferably about 2-10°C
  • the two surfaces which contact the aqueous solution are optically clear, so that the nuclear lysis and chromatic-spreading processes can be observed by optical or epifluorescence microscopy.
  • the transverse fluid flow can be driven by any of several different physical processes, for example, evaporation from unsealed boundaries of the interstitial aqueous solution, wicking, capillary action, or applied hydrostatic pressure.
  • the flux of the liquid across the surface is preferably approximately 20nl/mm 2 /hour.
  • the fibers may be optionally crosslinked to the surface. For example, if one or both surfaces are poly(L- lysine)-coated, then crosslinking may be achieved with using formaldehyde or UV irradiation.
  • the chromatin fibers may be continued to be incubated in order to cause spontaneous, periodic cleavage of the DNA within them, by means of residual apopotic machinery from the cell-lysate. This spontaneous cleavage leads to the production of ordered 1-dimensional and 2-dimensional arrays of chromatin fragments or DNA- containing beads.
  • the present invention also provides a method for preparing fragmented chromatin comprising (a) lysing cells in aqueous solution; (b) lysing the nuclei of the cells between two surfaces which are separated by an aqueous layer; (c) allowing transverse fluid flow to occur between the surfaces to release the chromatin fibers, which then attach to one or both surfaces; and (d) incubating the chromatin for a period of time sufficient for the spontaneous cleavage of the DNA within the chromatin.
  • the chromatin fibers are preferably incubated at 2-10°C for at least 24 hours, to produce spontaneous chromatin fragmentation.
  • the fragmented chromatin is likely produced through the activation of endogenous nucleases, which most likely are components of the residual apopotic machinery of the lysed cells. These nucleases operate on the spread chromatin to convert it into an ordered one- or two-dimensional array of chromatin fragments or DNA-containing beads.
  • This spontaneous fragmentation can be modulated or blocked by many methods including: a transient pulse of heat and phosphatase; by EDTA or EGTA; or by inhibition of endogenous caspases, for example by addition of the peptides Ac-YVAD-CHO or Ac-DEVD-CHO that are competitive inhibitors of caspases.
  • the fragmentation of the chromatin may be achieved by perfusing or diffusing into the interstitial aqueous solution an agent to effect a cleavage of DNA strands.
  • an agent for example, a deoxyribonuclease enzyme would be such an agent.
  • the present invention further provides a method for preparing fragmented chromatin comprising (a) lysing cells in an aqueous solution; (b) lysing the nuclei of the cells between two surfaces which are separated by an aqueous layer; (c) allowing transverse fluid flow to occur, to release the chromatin which attaches to one or both surfaces; and (d) perfusing in an agent which cleaves the chromatin to produce fragments.
  • the fragments of chromatin or DNA-containing beads produced according to the above method can be induced to detach from an underlying substrate or matrix. Once detached, the beads diffuse freely in solution. The diffusion can be tracked by means of an X-Y projection, and translational diffusion co-efficients can be calculated.
  • the beads when raised in temperature from about 2-10°C to about 15-37°C exhibit a transition from solid to liquid-crystalline form. The transition from solid to liquid-crystalline form has not been previously described for chromatin.
  • the present invention also provides a method for preparing liquid-crystalline chromatin comprising (a) lysing cells in an aqueous solution; (b) lysing the nuclei of the cells between two surfaces which are separated by an aqueous layer; (c) allowing transverse fluid flow to occur, to release the chromatin which attaches to one or both surfaces; (d) optionally perfusing in an agent which cleaves the chromatin to produce fragments; (e) incubating the chromatin for a period of time sufficient for cleavage of the chromatin to produce fragments; and (f) heating the chromatin to a temperature sufficient to allow a solid to liquid crystal transition to occur in the chromatin fragments.
  • the temperature is preferably raised from about 2-10°C to at least 15°C, more preferably to a temperature ranging from about 15°C to about 37°C Applications of the Invention
  • the chromatin prepared according to the present invention offers significant advantages over chromatin prepared according to the methods of the prior art.
  • the yield of the chromatin prepared by the present invention is 10- to 20-fold higher than with prior art methods.
  • the method of the invention allows the preparation of a wide variety of chromatin fibers, spanning from the interphase to metaphase levels of condensation.
  • the chromatin prepared according to the present invention is prepared within the lysate from cells which were alive at the start of the procedure; therefore many metabolic functions of the cell are preserved during the nuclear lysis and chromatin- spreading processes. This allows for the study or utilization of various biochemical functions of the cells, and in particular the apopotic machinery of the cells. Due to the numerous advantages of the method of the present invention, the chromatin fibres and beads prepared according to the method of the present invention can be used in a wide variety of techniques, some of which are outlined below.
  • the chromatin prepared according to the method of the present invention can be used as a substrate for physical genome mapping, in conjunction with sequence-specific hybridization probes.
  • chromatin prepared in accordance with the present invention can be used as a substrate for the in-situ hybridization of nucleic acid probes, for example in a FISH (fluorescence in situ hybridization) assay.
  • chromatin prepared according to the present invention can be used as a tool to investigate the natural organization of chromatin within cells, and to investigate changes in this organization which result from the actions of drugs or other active compounds on the cells.
  • chromatin prepared in accordance with the present invention is obtained in a relatively intact state. Histones and non-histone chromosomal proteins remain associated with this chromatin, and large-scale structures above the nucleosomal (3-10 nm) level remain appreciably intact.
  • chromatin prepared in accordance with the present invention can be used as a tool to investigate normal or aberrant apoptosis in cells.
  • Such cells may include normal cells, disease-associated cells, and cells that have been treated with various types of agents, including therapeutic agents and agents which induce or inhibit apoptosis.
  • the spread chromatin is uniformly stained, for example with the DNA- specific dye Hoechst 33258, and is then examined under the epifluorescence microscope, to detect and quantitate the pattern of cleavage of the chromatin by endogenous nucleases which are activated by means of residual apopotic machinery from the lysed cells. Conditions which influence the operation of the apopotic machinery will result in different chromatin cleavage patterns.
  • chromatin in this assay can be utilized as a functional screen for alterations or defects in the underlying biochemical and genetic pathway which control apoptosis (Rowan et al., 1996; Korsmeyer, et al., 1993; Camplejohn, R.S. et al., 1995; Cosulich et al., 1996; Miyashita, et al, 1994).
  • the DNA within chromatin fragments or beads prepared by the method of this invention can be isolated, cloned, and further characterized.
  • single beads may be removed individually from an optically-clear surface using a micromanipulated micropipette. Segments of DNA within the beads can be amplified using PCR, cloned, and sequenced using techniques known in the art.
  • the chromatin fragmentation assay as described in the present invention, could be employed as an early-stage test within a hierarchical screening procedure, for the rapid detection of functional aberrations in the biochemical and genetic pathways which control apoptosis.
  • Defective operation of an apoptosis pathway may reveal underlying defects in the participating proteins and enzymes, which may in turn result from mutations in the underlying genes.
  • Such a hierarchical testing scheme could rapidly restrict and delimit a set of candidate apoptosis-controlling genes, to be passed on to a gene-sequencing phase of the hierarchical screening procedure.
  • Hierarchical screening methods which culminate in DNA-sequencing tests are described in US patent #5,545,527 issued to Stevens, J.K. and Dunn, J.M. (August 13, 1996).
  • Apoptosis-controlling genes are of broad interest in human health and disease, because they are known or predicted to have oncogenic or tumor-suppressive properties.
  • chromatin prepared in accordance with the present invention can be used to evaluate weak points in DNA, for example weak points associated with fragile chromosome syndromes, such as the Fragile X syndrome.
  • weak points in the DNA can be inferred from fragmentation of chromatin spreads prior to, or in the absence of, or after an experimental inhibition of, the usual spontaneous fragmentation due to the residual apopotic machinery of the cell.
  • the sequences surrounding the break points can be evaluated, by means of physical isolation, cloning, and sequencing.
  • chromatin prepared in accordance with the present invention can be used for physical isolation of labelled or tagged chromosomal fragments, for example chromosomal fragments which specifically hybridize with a probe, or which lie adjacent to the site of probe-hybridization.
  • chromatin prepared in accordance with the present invention can be used as a substrate for hybridization with a pair of such adjacent probes, which represent two genetic markers which flank a mutation producing a detectable phenotype, for example a disease-related phenotype. Chromatin prepared in accordance with the present invention accordingly can be used to obtain a genomic fragment which lies between two such probes or genetic markers. This genomic fragment is a useful intermediate in an experimental strategy that is directed toward the positional cloning of a disease- related mutation.
  • a highly-ordered series of clone "microlibraries” can be generated to cover, with a minimum of redundancy, a large genomic subregion, or perhaps even the entire genome, of an arbitrary animal species, and perhaps of an arbitrary eukaryotic species. This can be achieved by spreading chromatin on a surface, allowing or inducing fragmentation to produce an ordered set of chromatin fragments, sequentially isolating the ordered fragments, for example with a micromanipulated micropipette, and applying a PCR/cloning strategy to these isolated fragments.
  • the method can be used to prepare a collection of highly compact chromatin particles, for use in testing or calibrating a matrix for transport- mediated separation of macromolecules.
  • a transport-mediated separation process is electrophoresis.
  • matrixes include microlithographic arrays with uniform spacing of posts and pores; and arrays with randomly-spaced posts and pores.
  • the present invention provides a method to allow observation of solid - liquid-crystalline transitions within chromatin. Indeed, prior to this invention, there was a lack of means to produce chromatin entities which could undergo the solid /liquid crystal transition. The transition is well known in DNA, but not in chromatin.
  • liquid-crystalline chromatin particles prepared according to the method of this invention, and deposited in a 2-dimensional array on a suitable surface, might also find uses in practical applications, for example as component parts within chemical sensor arrays.
  • the present invention provides a method for preparing individual fibres of chromatin, and also webs and bundles of chromatin fibres, which display highly intricate, quasi-periodic or periodic substructures.
  • Sources of Chemical and Biological Reagents Water is obtained from a Millipore Milli-Q system and stored frozen until use. Sea-salts for aquaculture are "scientific grade" from Coralife (Torrance, CA). All other buffers and salts are ACS reagent grade or better. EDTA, EGTA, formaldehyde, poly(L-lysine), and Hoechst 33258 are from Sigma. A mouse monoclonal anti(histone HI), is product # MAB1276 from Chemicon (Temecula, CA). Ac-DEVD-CHO, Ac-YVAD-CHO and Ac-YVAD-CMK are from Bachem (King of Prussia, PA).
  • DNAase I (amplification grade), TdT, and biotin-14-dCTP were from Gibco/BRL.
  • RNAase TI are from Sigma.
  • Rhodamine-conjugated goat anti(mouse IgG) fluorophore:protein molar ratio 2.7
  • Nanogold (20 nm)-conjugated streptavidin is from Sigma.
  • Rhodamine-conjugated anti(biotin) and Texas Red-conjugated anti(biotin) are from Jackson ImmunoResearch (West Grove, PA).
  • Teflon-coated slides with 5 mm diameter glass-bottom wells (Cel-Line, Newfield, NJ) are dipped in fresh aqueous 0.01% poly(L-lysine) at 24° C for 10 minutes. They are then air-dried at 60° C for 60 minutes and allowed to cool to room temperature. The coated slides are examined under brightfield and epifluorescence microscopy at 100 X magnification for surface defects and background fluorescence, and bad slides are rejected. Slides are always used within several hours of being made. Other protocols for preparation of poly(L-lysine)-coated slides, which differ somewhat from the above method, can also be used. (Meng, X. et al., 1995). It will be appreciated that other types of flat surfaces besides poly(L-lysine)-coated glass, could also be used, as discussed in Example 3 below. 4. Procedure for Making Chromatin Spreads.
  • Zebrafish embryos are allowed to develop normally to 75% epiboly (approximately 8 hours post-fertilization). At this developmental stage, they contain ⁇ 8,000 cells each. The embryos are dechorionated and deyolked manually in glass depression slides which contain "staining solution" (l ⁇ g/ml Hoechst 33258 in either Milli- Q water or Milli-Q water supplemented with 60 mg/L sea-salts).
  • the "animal caps" which result from this deyolking process (Sagerstrom et al., 1996) are transferred to individual wells of a ⁇ oly(L-lysine)-coated slide, along with a small volume of the staining solution. At this point additional chemicals, such as a 50 ⁇ M EDTA or EGTA solution, could be added to treat the animal caps.
  • Prefabricated 4 mm diameter #1 coverslips (Chase Instruments Corp., Norcross, Georgia) are placed over the individual animal caps. The weight of the coverslips cause the cells within the animal caps to lyse.
  • the slides are placed on a wet blotter paper in a covered Petri dish and incubated at 2-10°C for 24-72 hours.
  • nuclei lyse, and also a slow transferase fluid-flow occurs to spread the contents of the lysed nuclei gently across the poly(L-lysine)-coated surface.
  • the inventors have found that if the slides are incubated at 24-37°C then the nuclei do not lyse. It would appear that an early exposure to high temperature blocks or inhibits some heat-labile biochemical process which is required for lysis of the nuclei.
  • the rate of fluid-flow during the incubation can be estimated, based on the observation that, in this example, it appears to be driven by evaporation from the edges of the coverslip. If a well is 5 mm in diameter and a lOO ⁇ m space exists between the bottom of the well and the overlying coverslip, then this space will have a volume of 2.5 ⁇ l and an exposed surface area of 1.6 mm 2 . If 72 hours are required for evaporation to a semi-dry state, then the flux of liquid across the exposed surface area of a well is -20 nl/mm 2 /hour. In example 2, methods are described by which this rate of transverse fluid flow may be altered and controlled.
  • Lysine-coated Cel Line slide are placed in water + 60 mg/L sea-salts + 1 ⁇ g/ml Hoechst 33258. Upon lysis, the Hoechst 33258 dye spontaneously binds to the DNA, to render it fluorescent. Very little background fluorescence is observed.
  • Figures 1A and 2A show photomicrographs of spreads which have been stained by means of Hoechst 33258.
  • Figures IB and 2B show digital representations of periodic features from these respective photomicrographs. 6. Epifluorescence Microscopy.
  • Chromatin spreads can be imaged, at or near the Rayleigh limit of resolution, with epifluorescence microscopy using a Zeiss fixed-stage Axioskop mounted on a pneumatic air-table (TMC, Peabody, MA).
  • TMC pneumatic air-table
  • a 50 watt Hg source is filtered through a Shott BG38 bandpass filter (300 nm ⁇ ⁇ ⁇ 715 nm).
  • illumination sources for example lasers, and other combinations of microscope objectives and secondary lenses, may also be used with comparable results.
  • Photomicrography, Direct Electronic Imaging, and Screen Projections can be taken with a Zeiss MC-80 magnetic-shutter 35-mm camera, using 100 ASA or 400 ASA Ilford Delta professional-grade black/white film. In some cases it is advantageous to use a total magnification of 2,000X, to spread the image over a large area of a 35-mm film negative, so that the silver grain density of the film negative is not a factor which limits the spatial resolution. Exposures >1 min long should be avoided, as these tend to be slightly blurred, probably from small vibrations which passed through the pneumatic air-table. Films are developed with Ilfotec HC developer (Ilford, Cheshire, UK) under the "maximum resolution" protocol specified by the manufacturer.
  • Negatives are either printed by hand on Ilford Multigrade IV glossy paper, or are digitized with a Nikon IS-1000 slide scanner (8 ⁇ m pixel size). It will be appreciated that other photographic cameras, films, developing protocols, printing protocols, and digitizing instruments may also be used, with comparable results.
  • chromatin spreads at moderate resolution by means of a CCD camera, which fits on a microscope port by means of a standard adaptor. Acceptable resolution may be achieved with a 1,000,000 pixel CCD camera. A CCD camera having fewer pixels may also be used, although this will have lower resolution than the 1,000,000 pixel CCD camera or the photographic film.
  • the aqueous interstitial layer between the two surfaces may be unsealed, and therefore exposed to the atmosphere Spreadmg of chromatin fibers will m this case occur by means of evaporation-driven fluid flow
  • the rate of evaporation can be controlled by manipulation of the humidity of the atmosphere
  • the aqueous interstitial layer between the two surfaces may be unsealed, and intentionally placed into external contact with a "wick", constructed for example from cellulose fibre
  • the wick will absorb water from the aqueous interstitial layer, thereby effectmg a fluid-flow between the two surfaces
  • the rate of fluid flow can be controlled by manipulating the chemical or physical composition of the wick
  • the aqueous interstitial layer between the two surfaces may be unsealed, and intentionally placed mto external contact with a capillary tube or with a pair of surfaces bonding an air space This contact will effect fluid flow, driven by capillary forces
  • the rate of fluid flow can be controlled by manipulating the chemical or physical composition of the surfaces which exert the capillary forces 4. Hydrostatic Pressure.
  • the aqueous interstitial layer between the two surfaces may be unsealed, and intentionally placed mto contact with a source of new aqueous fluid Flow of fluid from this source mto the aqueous interstitial layer will produce a consequent flow across the surfaces
  • the rate of fluid flow can be controlled by manipulating the hydrostatic pressure of the external fluid source
  • exogenous agents for example nucleases or apoptosis inhibitors, may be added to the external fluid source, thereby becoming transferred into the aqueous interstitial layer, and commg mto contact with the spread chromatin
  • exogenous agents for example nucleases or apoptosis inhibitors
  • Zebrafish embryos are used to prepare Hoechst 33258-stained nuclear spreads according to the procedure described in Example 1.
  • the slides (with coverslips) are incubated in the dark in a humidified Petri dish for 24 hours at 4°C
  • the coverslips are then carefully removed with a fine forceps.
  • the slides are then dried in the dark in a nonhumidified Petri dish for 24 hours at 4°C It appears that this drying step causes the spreads to adhere more tightly to the poly(L-lysine)-coated surface.
  • the spreads are then rehydrated by adding 20 ⁇ l of water to each 5-mm diameter well and incubating for 10 min at 24°C
  • 4% formaldehyde may be added to the water during the rehydration step, to chemically crosslink the histones or DNA within the fibers to the poly(L-lysine) surface (Jackson, 1978; Ohba et al., 1979; Solomon & Varshavsky, 1985; Kuykendall & Bogdanffy, 1992, 1994).
  • the water or formaldehyde solution is then removed.
  • the DNA in the spread fibres is then nicked, and the RNA is destroyed, by adding to each well 20 ⁇ l of 0.01 U/ ⁇ l DNAase I + 1 U/ ⁇ l RNAse TI in 10 mM MgCl 2 , 50 mM Tris-HCl (pH 7.5).
  • the slides are incubated in the dark in a humidified petri dish for 20 min at 37°C This is followed by a wash step with 20 ⁇ l of 50 mM Tris-HCl (pH 7.5) per well. Care is required at this step to thoroughly inactivate or remove the DNAase I, otherwise the spreads will degrade during subsequent steps.
  • 0.5°C Embryos are maintained in "fish water” until the dome to sphere stage. They are then dechorionated, and placed in agarose-coated Petri dishes containing Ringer's solution. Next, Br-dU is added to the living embryos, to a final concentration of 10 ⁇ M, in water + 60 mg/L sea salts + 2% DMSO. The embryos are allowed to develop for 3 hr. Optionally, 0.5 ⁇ g/ml nocodazole can be added at this stage, and the embryos are incubated an additional 30 min, to produce a cell-cycle arrest at early pro-metaphase.
  • Figure 13 presents an image of a representative interphase nucleus from an embryo which has been incubated in Br-dU, and then stained with an anti (Br-dU) antibody, followed by (TRITC)-conjugated anti (IgG).
  • This image is a superimposition of 8 slices from a confocal microscope. Each confocal slice was 20 nm thick, and the slices were separated by 1 ⁇ m. The confocal slices were collected with a 100X oil-immersion plan-apochromat objective. This figure reveals that the cells of an embryo are able to successfully incorporate Br-dU into their replicating DNA.
  • chromatin spreads are prepared in the usual way, and counterstained with Hoechst 33258. The spreads are subjected to a brief crosslinking (10 min treatment with 4% formaldehyde), to bind the spreads to the poly(L-lysine) surface.
  • confocal microscopy is to be used to examine nuclear spreads, then it is advantageous to not use a dye which emits blue light. This is because most confocal microscopes are not equipped with UV lasers. It is better to use a DNA dye which can be stimulated by visible light. Examples of preferred dyes include the TOTO series of dimeric cyanine dyes, with fluorescence excitation and emission spectra which span the visible and near-infrared region. However, these dyes have significant affinity for RNA, as well as DNA. Thus, it is necessary to treat spreads with RNAase, before applying these dyes. (See Haughland, R. P., 1996) 6. Imaging of Spreads with Confocal Microscopy.
  • TRITC rhodamine
  • the 568 nm laser line is used for excitation, and a detector wavelength of 590 nm is selected by means of a dichroic filter.
  • a long probe (>100 nucleotides in length) can be prepared by nick-translation with biotin-dUTP or digoxygenin-dUTP.
  • a short probe ( ⁇ 100 nucleotides in length) can be prepared by addition of biotinylated nucleotides to the 3'-OH terminus of the probe, through the action of a terminal deoxynucleotidyl transferase enzyme.
  • biotinylated nucleotides can be directed monitored by labelling the 5' terminus of the probe with ⁇ -[ 32 P] ATP, using T4 polynucleotide kinase, followed by removal of unincorporated ⁇ -[ 32 P] ATP, followed by examination on a 15% polyacrylamide gel, followed by autoradiography.
  • the spread is pre-hybridized in 70% formamide, in the presence of a "blocking" agent such as BSA or denatured random-sequence DNA.
  • a blocking agent such as BSA or denatured random-sequence DNA.
  • Spreads are made according to the procedure described in Example 1. The inventors have discovered that if the spreads are kept in a hydrated state, at a temperature of about 2-10°C, for a period of >24 hours, and preferably for a period of 24-72 hours, then they will spontaneously fragment to produce arrays of chromatin "beads" which display significant 1- or 2-dimensional order. Examples of such fragmented spreads are shown in Figure 5. The spontaneous fragmentation of nuclear spreads appears to occur through the operation of residual apopotic machinery, which originates from the lysed cells.
  • Zebrafish embryos at 7 hr post-fertilization are incubated 30 min in 0.5 ⁇ g/ml nocodazole. They are then dechorionated, placed individually in wells of a poly(L- lysine)-coated Cell Line slide, in a buffer containing 1 ⁇ g/ml Hoechst 33258, and subjected to the lysis and spreading procedure at 2-10°C, as outlined in example 1 above.
  • the inventors have found that the spontaneous fragmentation of chromatin spreads can be partially or completely inhibited by perfusion of Ac-YVAD-CHO or Ac- DEVD-CHO peptides into the media which bathes the chromatin spreads.
  • These peptides are highly specific competitive inhibitors of caspases (cysteine-aspartyl-specific proteinases).
  • the Ac-YVAD-CHO and Ac-YVAD-CMK peptides are competitive inhibitors of caspases-1,4,5, and the Ac-DEVD-CHO peptide is a competitive inhibitor of caspases-2,3,7 (Margolin et al., 1997; Talanian et al., 1997; Thornberry et al., 1997).
  • Caspases are required for the execution phase of apoptosis. Therefore, the blocking of fragmentation of spreads with Ac-YVAD-CHO or AC-DEVD-CHO peptides, serves as a model for the predicted phenotype of a mutation which inactivates the protein product of a gene encoding a caspase.
  • the spread chromatin prepared according to the present invention could be used within a hierarchical testing scheme, for rapid detection of inactivating mutations in genes which are required for the execution phase of apoptosis. Hierarchical screening methods which culminate in DNA-sequencing tests are described in U.S. Patent No. 5,545,527, issued to Stevens, J.K. and Dunn, J.M. (August 13, 1996).
  • the method can be summarized as follows. At 5 hours post-fertilization, zebrafish embryos are placed in water + 60 mg/L sea-salts + 250 ⁇ M Ac-DEVD-CHO, Ac- YVAD-CHO or Ac-YVAD-CMK. Embryos are incubated in solutions of the individual inhibitors at 28°C for 60 minutes. The embryos are then used to prepare chromatin spreads according to our standard protocol (Example 1 above) except for the following modification. Just before the addition of the coverslip, 40 ⁇ l of the appropriate caspase inhibitor solution is added to each well of the slide, which contained a single animal cap.
  • the inventors have found that the fragmentation process can be partially or completely inhibited by adding EDTA or EGTA for the aqueous solution which bathes the chromatin spreads.
  • This is a model for the predicted phenotype of an inactivating mutation in a gene for a nuclease which fragments the chromatin during apoptosis, or in a gene for a DFF-like factor which activates this nuclease during apoptosis. (Liu, X., 1997.)
  • Example 9 Sensitive Detection of the Fragmentation of Chromatin. as Induced by Treatment with Chemotherapeutic Agents
  • the present invention provides an extremely sensitive way to assess whether cells are sensitive or resistant to apoptosis-inducing chemotherapeutic agents.
  • a useful application of this test would be to evaluate tumor biopsies. Tumors are often characterized as being deficient in normal apoptotic responses. For example, one may test for resistance to induction of apoptosis by campothecin, which is a potent inhibitor of topoisomerase I and a widely-used chemotherapeutic agent. Treatment of normal cells with campothecin produces a very distinct and recognizable fragmentation phenotype, when examined in the format of chromatin spreads.
  • Figure 8A shows the fragmentation of nuclear spreads during campthothecin induced apoptosis.
  • One route of resistance to campothecin chemotherapy is believed to be due to an impairment of the apoptosis program; such an impairment should easily be recognizable in the spread chromatin assay.
  • the image can be digitized, a line-profile can be drawn along a trajectory of DNA-containing beads, and the resultant file of data points (pixel intensity I(x) versus position x) can be fit by least-squares methods to a single-component sine curve specified by the following formula:
  • the diameter of an individual bead can be measured, and the X-Y projection of the diffusion path of its center of mass can be tracked in 15-30 sec time-steps, by tracing onto a Mylar transparency affixed to a greyscale monitor.
  • the ambient temperature on the slide can be measured with a "pancake" thermistor probe (Omega Engineering, Stamford, CT).
  • the diffusion path of an individual bead can be analyzed with the following formula:
  • A. Colloidal Inert Particles Randomly-arranged wells in a glass surface, for example a soda-lime glass microscope slide, can be created in the following manner. First, a light coating of "kiln wash” (1 part kaolin, 1 part alumina hydrate, 1/2 part 400 mesh flint) is brushed and air-dried onto a flat ceramic shelf. Next, the glass slide is laid on top of the ceramic shelf, and heated to approximately 1,400 °F, according to a "fuse to stick" protocol. The wells are generated in the glass as negative-impression replicas of the colloidal inert particles in the kiln wash.
  • "kiln wash” (1 part kaolin, 1 part alumina hydrate, 1/2 part 400 mesh flint
  • the wells are generated in the glass as negative-impression replicas of the colloidal inert particles in the kiln wash.
  • the colloidal particles do not adhere to the glass, and therefore can be brushed, washed, or sand-blasted away, to produce wells.
  • this well-containing glass surface can be processed further, e.g. by coating with a surface layer, such as poly(L-lysine), which has affinity from DNA and chromatin.
  • a surface layer such as poly(L-lysine)
  • Raised obstacles on a glass surface can be produced by heating a flat soda-lime glass microscope slide above the liquidus temperature (1400 °F), and then slowly cooling through the vitrification point ( ⁇ 1350 °F). During the cooling process, vitrification crystals form on the surface of the glass.
  • the inventors have determined that often the vitrification crystals form a quasi 2-dimensional array. Microscopic examination at lOOx total magnification shows that the vitrification crystals are rectangular with ⁇ 5 ⁇ m width, and 5-20 ⁇ m length.
  • the presence of such raised obstacles, on one or both surfaces of an assemblage could comprise an "obstacle course" which presents barriers to the physical transport of macromolecules such as chromatin beads, e.g., during electrophoresis.
  • C. Glass Enamel An alternative method to produce raised obstacles is by applying a thin coating of glass enamel by means of a fine-point airbrush.
  • Glass enamel consists of colloidal glass particles in a volatile solvent. After applying the glass enamel to a soda-lime microscope slide, the slide is then heated under a "fuse to stick" heating schedule (to ⁇ 1350 °F) to cause the colloidal glass particles to bind to the substrate.
  • the presence of such obstacles could present barriers to physical transport of chromatin beads, e.g. during electrophoresis, as suggested above. See Lundstrom, B. (1983) for general protocols of glass fusing technology, which can be consulted for protocols to aid in the preparation of surfaces such as these.
  • Chromatin Fragments, or DNA- containing Beads by means of Micromanipulated Micropipette
  • the inventors have determined that, in chromatin spreads prepared according to the present invention, single chromosomes, and also DNA-containing chromatin beads can be visualized, using Hoechst staining, under a 40X (NA 0.75) objective, with 10-20
  • the working distance of the objective must be sufficient to allow manipulation of a micropipette, in the space between the objective and the sample.
  • a cooled CCD camera with >5megaHz transfer rate can be used.
  • an image-intensifying camera, with higher gain but somewhat lower resolution can be employed instead.
  • a chromatin spread can be prepared on an optical surface, and allowed to spontaneously fragment.
  • the overlying second surface for example a coverslip, can then be carefully removed with a fine forceps.
  • a micropipette guided by a micromanipulator, can be used to remove a single fragmented bead. For example, one can remove a bead which lies between two hybridization probes which are bound sequence-specifically to sites which span a disease- causing genetic locus.
  • Hiriyanna KT Varkey J, Beer M, Benbow RM (1988) Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo. J Cell Biol 107: 33-44. Jackson, V (1978) Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent. Cell 15: 945-954.
  • Senger G Jones TA, Fidlerova H, Sanseau P, Trowsdale J, Duff M, Sheer D; "Released chromatin: linearized DNA for high resolution fluorescence in situ hybridization”; Hum Mol Genet 1994 Aug;3(8):1275-1280 Sikorav JL, Pelta J & Livolant F (October 1994) "A liquid crystalline phase in spermidine- condensed DNA.” Biophys. J. 67(4), 1387-1392.
  • Genomic sequence sampling a strategy for high resolution sequence-based physical mapping of complex genomes. Nature Genetics 7, 40-47.

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Abstract

L'invention se rapporte à un procédé nouveau de préparation de chromatine à partir de cellules eucaryotes. Ce procédé est utile dans de nombreuses analyses, notamment dans les cas suivants: (1) établissement physique d'une carte de gènes par hybridation in situ; (2) essais pratiqués afin de détecter des fonctions défectueuses du mécanisme de l'apoptose dans des cellules; (3) obtention de fibres de chromatine fortement orientées, qui sont destinées à être utilisées dans des études de la structure de la chromatine en interphase ou en métaphase; (4) détection de sites fragiles dans les chromosomes de cellules normales, traitées chimiquement ou génétiquement mutantes; (5) préparation de réserves ordonnées de clones à partir d'un génome animal ou, éventuellement, d'un génome eukaryote; (6) obtention de fibres de chromatine et de corps de chromatine condensés, en plusieurs compacités et tailles, qui sont destinées à être utilisés dans des études des processus de transport des molécules, dont l'électrophorèse, et pour le calibrage de matrices de séparation de molécules; (7) obtention de corps de chromatine condensés, en plusieurs tailles, destinés à être utilisés dans des études portant sur les transitions cristallines solide-liquide de la chromatine et dans la préparation d'éléments détecteurs de cristalline liquide.
PCT/CA1998/000251 1997-03-18 1998-03-18 Procede de preparation de chromatine WO1998041651A1 (fr)

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CN103827322A (zh) * 2011-09-26 2014-05-28 凯杰有限公司 细胞外核酸的稳定化和分离
US9411930B2 (en) 2013-02-01 2016-08-09 The Regents Of The University Of California Methods for genome assembly and haplotype phasing
US9715573B2 (en) 2015-02-17 2017-07-25 Dovetail Genomics, Llc Nucleic acid sequence assembly
US10089437B2 (en) 2013-02-01 2018-10-02 The Regents Of The University Of California Methods for genome assembly and haplotype phasing
US10144952B2 (en) 2013-03-18 2018-12-04 Qiagen Gmbh Stabilization and isolation of extracellular nucleic acids
US10457934B2 (en) 2015-10-19 2019-10-29 Dovetail Genomics, Llc Methods for genome assembly, haplotype phasing, and target independent nucleic acid detection
US10526641B2 (en) 2014-08-01 2020-01-07 Dovetail Genomics, Llc Tagging nucleic acids for sequence assembly
US10724074B2 (en) 2012-09-25 2020-07-28 Qiagen Gmbh Stabilisation of biological samples
US10947579B2 (en) 2016-05-13 2021-03-16 Dovetail Genomics, Llc Recovering long-range linkage information from preserved samples
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US11203777B2 (en) 2015-11-20 2021-12-21 Qiagen Gmbh Method of preparing sterilized compositions for stabilization of extracellular nucleic acids
US11525155B2 (en) 2013-03-18 2022-12-13 Qiagen Gmbh Stabilisation of biological samples
WO2023172142A1 (fr) 2022-03-10 2023-09-14 Erasmus University Medical Center Rotterdam Procédés, compositions et dispositifs pour l'étalement de fibres de chromatine
US11807896B2 (en) 2015-03-26 2023-11-07 Dovetail Genomics, Llc Physical linkage preservation in DNA storage

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JP2014528719A (ja) * 2011-09-26 2014-10-30 プレアナリティクス ゲゼルシャフト ミット ベシュレンクテル ハフツング 細胞外核酸の安定化および単離
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