WO1998041651A1 - Method for preparing chromatin - Google Patents

Abstract

A novel method for preparing chromatin from eukaryotic cells is described. The method is useful in many assays, including: (1) physical mapping of genes by in situ hybridization; (2) testing for defective functions of the apoptosis machinery within cells; (3) obtaining highly-oriented chromatin fibres for use in subsequent studies on interphase or metaphase chromatin structure; (4) detecting fragile sites in the chromosomes of normal, chemically-treated, or genetically mutant cells; (5) preparing ordered pools of clones from an animal genome or perhaps from any eukaryotic genome; (6) obtaining chromatin fibres and condensed chromatin bodies, in a range of packing densities and sizes, for subsequent use in studies on molecular transport processes including electrophoresis, and for calibration of molecular separation matrixes; (7) obtaining condensed chromatin bodies, with a range of sizes, for subsquent use in studies on the solid-liquid crystal transitions of chromatin, and in preparation of liquid crystalline sensing elements.

Description

Title: METHOD FOR PREPARING CHROMATIN FIELD OF THE INVENTION

This invention describes a novel method for preparing spreads of eukaryotic chromatin, for a plurality of uses. BACKGROUND OF THE INVENTION

1. Mapping of Gene-Probes on Chromatin Spreads.

An important dimension of modern genome research involves mapping genes and unique DNA markers on the karyotype of a biological species. One way to determine gene and marker locations is with in situ hybridization. This involves making a "spread" of chromosomes, nuclei, or chromatin on a flat surface, and then hybridizing a DNA probe to the spread, in a sequence-specific fashion, with sequence-specificity conferred by Watson- Crick base-pairing (A:T, C:G pairing) between the probe and its complementary DNA sequence target within the spread.

Early low resolution methods for in-situ hybridization involved the hybridization of gene-probes to metaphase chromosome spreads. Hybridization signals were detected by autoradiography, or by the enzymatic production of an insoluble colored precipitate. (Sawin V.L. et al. 1978; Cannizzaro, L.A. & Emanuel B.S., 1984; Li C.B. et al,

1986; Brothman A.R. 1987; Bhatt, B. et al., 1988.)

Subsequent technical advances involved the detection of bound DNA probes by epifluorescence microscopy, and the development of methods for hybridizing gene probes to chromatin substrates which were less condensed than metaphase chromosomes.

One such method involved the hybridization of DNA probes against chromatin fiber spreads which had been derived from lysed interphase nuclei. Because the fibres were spatially extended, this afforded an improved spatial resolution. (Heng et al., 1992; Weigant J. et al., 1992; Lawrence, J.B. et al., 1992; Parra I & Windle B, 1993; Senger, G. et al., 1994; Heng HHQ & Tsui L-C, 1995; Michalet X et al., 1997.)

Some of the limitations for preparing chromatin spreads of the prior art include:

(1) 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.

(2) No single prior-art method can be used to obtain, in a controlled fashion, a range of fibres of different condensation states, spanning the continuum from interphase to metaphase levels of condensation. Thus, one class of prior-art methods produces prometaphase or metaphase spreads, while a different class of prior-art methods produces interphase spreads. In contrast, the present invention allows control over the extent of chromosome condensation, and therefore allows a range of chromatin structures to be produced.

(3) None of the prior-art techniques preserves the metabolic functions of the cell in an active form. In particular, the prior art techniques do not preserve the apopotic pathway, and therefore do not allow control over a spontaneous, ordered fragmentation of interphase or metaphase spreads, such as provided by the present invention.

(4) 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. In contrast, the present invention is general-purpose, and allows chromatin to be prepared for a multiplicity of uses.

2. In Vitro Apoptosis Systems.

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.)

Some of the limitations of prior art methods for studying apoptosis in vitro include the following: (1) A significant gap in measurement scales exists, between the lower limit of what can be observed cytologically, and the upper limit of what can be observed by gel electrophoresis, even using field-inversion techniques. Thus, many intermediates in the chromatin degradation process cannot be observed with prior art methods. In contrast, the present invention, by means of preparing apopotic nucleic and chromatin in a spatially dispersed form on flat optical surfaces, allows apopotic intermediates to be observed at or near the Rayleigh limit of resolution.

(2) 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. In contrast, 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.

3. Methods for Making Ordered Clone Pools.

The prior art has described methods for microdissection and cloning of specific chromosomal regions, for example regions which span a deletion or breakpoint. Prior-art methods have employed a mechanical or laser-directed microdissection of metaphase chromosomes, to achieve this goal. (Ludecke, H.J., 1989; Hadano S & Ikeda JE, 1993.) Some of the limitations of prior art methods for making clone libraries from chromosomal regions include:

(1) The use of metaphase chromosomes as the substrate for microdissection. This limits the spatial resolution of the microdissection procedure, and consequently implies that only relatively large (megabase-sized) regions can be physically isolated for microcloning. In contrast, the present invention uses chromatin spreads which are much more highly extended and dispersed. Thus, the minimum size of a microdissected region can be much lower.

(2) Significant physical force is required to break the chromosome, as a first step. In contrast, 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.

4. Preparation of Chromatin Samples for Electrophoresis and Other Transport Studies.

The prior art has sought to define experimental systems, which allow theoretical models of electrophoresis and other transport processes to be tested (Melenkevitz, J. and Muthukumar, M., 1990). For example, experimental systems have been designed which allow individual molecules to be tracked, in real time, during their electrophoretic movement. (Schwartz DC & Koval M., 1989; Smith, S.B. et al., 1989; Volkmuth WD & Austin RH, 1992; Volkmuth, W.D., 1995; Perkins, T.T. et al., 1995; Duke, T.A. et al., 1996; Yager, T.D. et al. (PCT Patent application #WO96/42012); Yager, T.D. et al. (PCT Patent application #WO96/42013)).

Some of the limitations of the prior art methods for preparing samples of DNA or chromatin for electrophoresis and other transport studies include the following:

(1) The prior art methods have not allowed the simultaneous isolation and examination of a range or variety of different DNA-containing objects. However, 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. (2) 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. However, the current invention proposes a method to allow the simultaneous tracking of a plurality or multiplicity of similar objects spanning a range of sizes. This would allow both the direct observation of individual objects (if this were desired); and also the generation of an "ensemble average" picture of the behaviour of the plurality or multiplicity of objects. The latter view would be useful, for example, for the rapid characterization of a new separation matrix for electrophoresis or some other transport process, such as could be constructed by microfabrication or glass- fusing routes. 5. Preparation of Chromatin Samples for Studies on the Solid-Liquid Crystal Transition.

A number of prior art studies have described solid-liquid crystal transitions in solutions of pure DNA, in the presence of various inorganic and organic salts. (Rill, R.L.,

1986; Brandes R & Kearns DR, 1986; Strzelecka, T.E. et al., 1988; Sikorav, J.L. et al., 1994.)

However, apparently, a solid-liquid crystalline transition has never before been described for a homogeneous preparation of chromatin.

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. (Livingston, G.K., 1980; Zhu, C. and Hieflje, CM., 1990; Tan, W. et al, 1992; Bronk, K.S. and Walt, D.R., 1994; Healey, B.G. et al., 1995; Healy, B.G. and Walt, D.R., 1997.) SUMMARY OF THE INVENTION

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). Preferably, 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. In a preferred embodiment, 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. Alternatively 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.

Accordingly, 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. To induce the spontaneous fragmentation, 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. Alternatively, 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 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; (9) to generate a highly- ordered series of clone "microlibraries" which require a minimum redundancy to span a large genomic subregion, or perhaps even the entire genome, of an arbitrary animal species, and perhaps of an arbitrary eukaryotic species; and (10) to prepare a collection of highly compact chromatin particles, for use in testing or calibrating a new matrix for separating the particles by means of a molecular transport process, for example electrophoresis.

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.

Accordingly, 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. Preferably, 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, prepared according to the method of this invention, comprise a unique system to allow the observation of solid - liquid crystal transitions within chromatin. In addition, when generated as a 2-dimensional array on a suitable surface, 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. (Seeman, N.C., 1982; Seeman, N.C., 1990; Zhang Y & Seeman NC, 1992; Seeman, N.C., 1996; Bethell D & Schiffrin DJ, 1996; Mirkin, CA. et al., 1996; Alivisatos, A.P. et al., 1996.) DESCRIPTION OF THE DRAWINGS

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).

DETAILED DESCRIPTION OF THE INVENTION Methods of the Invention

The present invention relates to a novel method for preparing chromatin. In particular, 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.

Any eukaryotic cells can be used in the method of the present invention. In one example, the cells may be derived from embryos of a multicellular eukaryotic organism. Preferably, 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 In a preferred embodiment, 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/mm²/hour. After the fibers have spread across the surface(s) by means of the transverse fluid flow, they 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.

Accordingly, 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. In one embodiment of the invention, 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.

Alternatively, 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. For example, a deoxyribonuclease enzyme would be such an agent. Accordingly, 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.

Accordingly, 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. For example, 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.

In one aspect, 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. In particular, 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.

In another aspect, 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. In this regard, 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.

In yet another aspect, 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. The detailed behaviour of chromatin in this assay, and particularly alterations or defects in the "phenotype" of chromatin fragmentation, 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). In another aspect, the DNA within chromatin fragments or beads prepared by the method of this invention can be isolated, cloned, and further characterized. In one example, 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.

In yet another aspect, 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.

In yet a further aspect, 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. The existence of 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. In yet a further aspect, 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.

In a further aspect, 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.

In a further aspect, 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.

In a further aspect, 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. An example of such a transport-mediated separation process is electrophoresis. Examples of such matrixes include microlithographic arrays with uniform spacing of posts and pores; and arrays with randomly-spaced posts and pores. Schwartz & Koval, 1989; Smith et al., 1989; Yager, T.D. et al. (PCT Patent application #WO96/42012); Yager, T.D. et al. (PCT Patent application #WO96/42013); Stevens JK & Ismailov AM,1996; Volkmuth & Austin, 1992; Volkmuth, et al., 1995; Duke, et al., 1996). In a further aspect, 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.

In a further aspect, 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. (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.) In a further aspect, 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. 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 assemblies. For example, a "molecular addressing" scheme can 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. (Seeman, N.C, 1982; Seeman, N.C, 1990; Zhang Y & Seeman NC, 1992; Seeman, N.C, 1996; Bethell D & Schiffrin DJ, 1996; Mirkrn, CA. et al., 1996; Alivisatos, A.P. et al., 1996.) The present invention is illustrated by the following examples. It is understood that these examples in no way limit the present invention and that the spirit of the invention extends to the entire area which is outlined above. EXAMPLES Example 1 Method of Preparing and Labelling Chromatin

1. 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) is from Sigma. Rhodamine-B-conjugated streptavidin (rhodamine /streptavidin molar ratio=4.9) is from Molecular Probes (Eugene, OR). 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).

2. Aquaculture and Embryology.

The procedure is described for embryos of the zebrafish (Danio rerio). It can be modified for other types of embryos, according to the appropriate methods described in the scientific or technical literature. Adult wild-type zebrafish are maintained using standard aquaculture methods (Westerfield, 1995). Sexes are pooled and housed in recirculating tanks of Milli-Q water supplemented with 60 mg/L sea-salts, pH 7-8, 27-28°C The fish are placed on a 14 h light/ 10 h dark cycle, and embryos are obtained by photo- induced spawning over clear glass marbles. For the first 10 cell-divisions, embryos from a single clutch typically are synchronous to within 1 cell cycle. Embryos are accurately staged by reference to Kimmel et al. (1995).

3. Preparation of Poly(L-Lysine)-Coated Slides.

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.

The procedure is described for cells from early zebrafish embryos although the method of the present invention can be applied to other cells from other biological sources. 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" (lμg/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 ρoly(L-lysine)-coated slide, along with a small volume of the staining solution. At this point additional chemicals, such as a 50 μM 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. During this time the 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 lOOμm space exists between the bottom of the well and the overlying coverslip, then this space will have a volume of 2.5μl and an exposed surface area of 1.6 mm². 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/mm²/hour. In example 2, methods are described by which this rate of transverse fluid flow may be altered and controlled.

5. Uniform Labelling of DNA in Spreads, by Means of Hoechst 33258. Deyolked "animal caps" are placed individually in wells of a poly(l-

Lysine)-coated Cel Line slide. They are placed in water + 60 mg/L sea-salts + 1 μg/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). For epifluoresence illumination, a 50 watt Hg source is filtered through a Shott BG38 bandpass filter (300 nm < λ < 715 nm). For detection of Hoechst 33258 staining, the light is then filtered through a Chroma #31000 dichroic filter set for blue fluorescence (λ_ex = 315-390 nm, λ_em = 435-490 nm). For detection of Texas Red or rhodamine staining, the light is instead filtered through a Zeiss #15 dichroic filter set for red fluorescence (λ_ex = 540-550 nm, λ_em > 590 nm). Samples are imaged with Zeiss 40X, NA=0.75 or 100X, NA=1.30 (oil immersion) plan-Neofluar objectives, in combination with 10-20X secondary magnification lenses. It will be appreciated that other illumination sources, for example lasers, and other combinations of microscope objectives and secondary lenses, may also be used with comparable results.

7. Photomicrography, Direct Electronic Imaging, and Screen Projections. Photomicrographs 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 (8μm 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.

Alternatively, it is possible to image the 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.

As yet another option, it is possible to project images on a screen, to which a gridded transparency has been affixed, for the purpose of tracing the motions of chromatin fragments in real time. This is useful for the purpose of monitoring the transport of fragments through the medium, for example by diffusion or electrophoresis, as discussed in Example 11 below. Example 2

Alternative Means to Effect Spreading of Chromatin Fibers

1. Evaporation from Edges of Solution.

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

2. Wtcktng From Edges of Solution.

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

3. Capillary Action. 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 This method of controlling the fluid-flow also has the additional advantage that 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 Example 3 Alternative Surfaces

Many alternative surfaces besides poly(L-lysιne)-coated glass could be employed in the chromatin-spreading procedure of the present invention The chief requirement appears to be that the two surfaces which bound the aqueous interstitial layer are approximately flat and parallel Alternative surfaces include, but are not limited to glass which is coated with polyacrylamide (Morozov et al , 1996), glass which is coated by a modified polyacrylamide such as poly-dimethyl-acrylamide (Timofeev et al , 1996), freshly-cleaved, glow-discharged, or H+-exchanged mica (Rivetti et al , 1996), monolayer silane films on substrates made of silica or silicon (Chπsey et al , 1996), or a variety of hydrophobic surfaces, to which DNA or chromatin may adhere in a pH-dependent fashion (Allemand et al., 1997). It will be appreciated that different surfaces may be selected according to the subsequent use which is envisioned. For example, if the spread chromatin is to be imaged by scanning-probe microscopy, then a mica surface would be preferred (Rivetti et al., 1996). Example 4 Alternative Labelling and Detection Procedures

1. Labelling of Nuclear Spreads with Anti(Histone HI).

Chromatin spreads are prepared according to the procedure described in Example 1. After 24 hours incubation in a humidified petri dish at 4°C, the coverslips are removed with a pair of fine forceps and the slides are allowed to dry. A wash is performed by adding 20 μl of PBS (pH = 7.2) to each well and incubating the slides in the dark in a humidified petri dish for 10 min at 24°C The wash is removed and a blocking step is then conducted by adding 20 μl of PBS (pH = 7.2) + 5% normal goat serum, and incubating as above. The blocking agent is removed, and 20 μl of ~0.5 μg/μl anti(Hl) in PBS is then added to each well. The samples are incubated in the dark in a humidified petri dish for 2 hours at 24°C This is followed by two five-minute washes with 20 μl of PBS (pH = 7.2) at 24°C in the dark. Next 20 μl of 0.07 μg/μl TRITC-conjugated goat anti(mouse IgG) in PBS is then added to each well. The samples are incubated in the dark in a humidified petri dish for 30 minutes at 24°C This is followed by two five-minute washes with 20 μl of PBS (pH=7.2) at 24°C in the dark. The chromatin spreads in each well are then restained with 20 μl of 4 μg/ml Hoechst 33258 in PBS. This restaining is done for 10 minutes at 24°C in a humidified petri dish in the dark. Approximately 18 μl of liquid is removed from each of the wells, and then the wells are covered individually with 4 mm diameter round coverslips. The spreads are viewed immediately under epifluorescence microscopy. Results are shown in Figure 3. The left panels in this figure show the staining with Hoechst 33258, while the right panels in this figure show the staining with anti (HI).

2. Nick-labelling of Spreads by TdT-mediated biotin-dCTP Incorporation.

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 μl of water to each 5-mm diameter well and incubating for 10 min at 24°C As an option, 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 μl of 0.01 U/μl DNAase I + 1 U/μl RNAse TI in 10 mM MgCl₂, 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 μl 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. This is achieved by adding to each well 20 μl of 4% formaldehyde and incubating for 10 minutes. This is followed by a second wash step in which 20 μl of 50 mM Tris-HCl (pH 7.5) is added per well. A modified TUNEL reaction is then conducted by adding to each well 20 μl of 0.3 U/μl TdT (terminal deoxynucleotidyl transferase) + 20 mM biotin-14-dCTP + 50 ng/μl BSA in 0.1 M potassium cacodylate (pH 7.2), 2 mM CoCl₂, 0.2 mM DTT. The slides are incubated in the dark for 30 min at 37°C in a humidified petri dish. The TUNEL reaction is stopped by adding to each well 0.8 μl of 0.5 M EDTA (pH 8.0) and incubating for 5 min. The EDTA solution is removed, and two wash steps are conducted with 20 μl of 50 mM Tris-HCl (pH 7.5) per well. Next 20 μl of either 10 ng/μl or 100 ng/ml streptavidin-rhodamine B (rhodamine/ streptavidin molar ratio = 4.9) is added to each well. The slides are incubated in the dark in a humidified petri dish for 60 min at 24°C This is followed by two wash steps with 20 μl of 50 mM Tris-HCl (pH 7.5) per well. Finally, round coverslips (4 mm diameter) are placed over each sample. Slides are viewed immediately under epifluorescence microscopy. Figure 4 presents examples of nuclear spreads which were treated in this fashion, to reveal the presence of DNA.

3. Labelling of spreads with colloidal gold, followed by imaging with DIC (Nomarski) Microscopy.

Spreads are nick-labelled by TdT-mediated biotin-dCTP incorporation. This is achieved according to the method described in section (2) of Example #4. However, in place of a fluorescently-labelled strepdavidin, colloidal gold-labelled streptavidin is applied. For example, the inventors have successfully employed a nanogold-conjugated streptavidin, in which the nanogold particle diameter -20 nm (Sigma Chemical Co., St. Louis, MO). After washing (as described in section (2) of example #4), the specifically- bound nanogold is detected by means of differential interference contrast (Nomarski) microscopy. (See DeBrabander, M. et al., 1993 and Hiriyanna et al., 1988).

4. Replication Banding by Br-dU Incorporation. (1) All operations involving live zebrafish embryos are conducted at 28.5 +

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 μM, in water + 60 mg/L sea salts + 2% DMSO. The embryos are allowed to develop for 3 hr. Optionally, 0.5 μg/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 μm. 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. (2) Next, 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.

(3) Next, the spreads are blocked with 5% goat serum. Then, anti(Br-dU) antibody is added in 5% goat serum, followed by a wash step, followed by addition of fluorescent secondary antibody, for example TRITC-conjugated anti (IgG). 5. Labelling of Spreads with Alternative DNA Dyes.

If 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.

The following procedure can be used for spreads which are immunohistochemically stained with a rhodamine (TRITC)-conjugated antibody (λex = 544 nm, λem= 572 nm). A Leica TCS4D confocal microscope can be used, which employs a Omnichrome krypton-argon ion laser illumination source and a 100X (NA = 1.4 oil immersion) plan-apochromat objective. 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 series of confocal slices, each 20 nm thick and spaced 1 mm apart, are collected at 512 X 512 pixel resolution, with 8X line-averaging. It will be appreciated that other fluorescent immunohistochemical stains, and other confocal microscopes can also be used with comparable results. 7. Preparation of Chromatin Spreads, and Sequence-Specific Hybridization of Nucleic Acid Probes.

(i) The chromatin spreading procedure is conducted in the presence of RNAase. This insures that all RNA will be destroyed, so that it will not interfere with subsequent steps. (ii) One of the two surfaces which bounds the aqueous interstitial layer, in this example a glass coverslip, is carefully removed by means of a fine forceps.

(iii) The spread is subjected to a brief formaldehyde fixation, to bind it to the poly(L- lysine) surface. As an option, 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.

(iv) The spread is denatrued by immersion in 70% formamide /2X SSC at 70°C for 2 min. This is followed by immersion in cold 70% ethanol (1 min), then in cold 90% ethanol (1 min), and finally in cold 100% ethanol (1 min).

(v) A long probe (>100 nucleotides in length) can be prepared by nick-translation with biotin-dUTP or digoxygenin-dUTP. Alternatively, 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. In the latter case, the addition of biotinylated nucleotides can be directed monitored by labelling the 5' terminus of the probe with γ-[³²P] ATP, using T4 polynucleotide kinase, followed by removal of unincorporated γ-[³²P] ATP, followed by examination on a 15% polyacrylamide gel, followed by autoradiography.

(vi) The spread is pre-hybridized in 70% formamide, in the presence of a "blocking" agent such as BSA or denatured random-sequence DNA.

(vii) This spread is hybridized to the probe in 70% formamide and dextran sulfate. (viii) Washing to remove unbound probe, followed by binding of fluorophore-conjugated anti(biotin) or anti (digoxygemin) antibody, followed by washing to remove unbound antibody. Special solvents, for example NaOH/ethanol or alkaline borate solution, may be employed to cause maximal repulsion between phosphates in the backbone of the DNA, thereby spreading the DNA out to a maximal extent. Yokota, H. et al., 1995; Senger, et al, 1994. Example 5 Spontaneous Fragmentation of Chromatin Spreads

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.

Example 6

Obtaining Spreads of Chromatin in the Metaphse and Pro-Metaphase Stages of Mitosis, in

Both Unfragmented and Fragmented States.

Zebrafish embryos at 7 hr post-fertilization are incubated 30 min in 0.5 μg/ml nocodazole. They are then dechorionated, placed individually in wells of a poly(L- lysine)-coated Cell Line slide, in a buffer containing 1 μg/ml Hoechst 33258, and subjected to the lysis and spreading procedure at 2-10°C, as outlined in example 1 above.

(1) After 24 hours of incubation, a large number of metaphase chromosomes, prometaphase chromosomes, and very early prometaphase chromosomes are observed in the spreads. Some of the chromosomes in the last-named category clearly consisted of pairs of daughter chromatids, which are joined at the centromere. In some cases, these are extended as far as 25 μm in length, which represents a 5-10X extension beyond what is normal for metaphase chromosomes. The top two panels of Figure 3 show an example of prometaphase chromosomes. (2) Typically, when observed after 60 hours, a number of the metaphase, prometaphase, and early prometaphase chromosomes are found to have undergone spontaneous fragmentation, to produce linearly-arranged series of DNA-containing beads. Thus implies that the method to generate ordered arrays of DNA-containing chromatin fragments applies also to metaphase and prometaphase chromosomes. This in turn suggests that linearly-arranged pools of genomic clones can be generated from metaphase or prometaphase chromosomes. Example 7 Block of Spontaneous Fragmentation of Spreads with Caspase Inhibitors

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. Generalizing this concept, 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 μM 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 μl of the appropriate caspase inhibitor solution is added to each well of the slide, which contained a single animal cap. This operation insures that the inhibitory peptide will be present at high concentration during the time when fragmentation of the spread ordinarily would occur. After a 5 minute incubation at 24°C, excess fluid is removed and the wells are covered individually with 4 mm diameter round coverslips. Slides are incubated in a humidified Petri dish at 4°C for 72 hours with Hoechst 33258 staining, and then examined by epifluorescence microscopy. Figures 9, 10 and 11 show examples of chromatin spreads which have been partially or completely blocked in their spontaneous fragmentation, through the application of the caspase inhibitor Ac- DEVD-CHO. Example 8 Block of Fragmentation with Nuclease Inhibitors

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. Example 10

Quantitative Analysis of Periodic Cleavage Patterns

To analyze a periodic fragmentation pattern in a nuclear spread, 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:

T2πx 1

I (x) = b + flrj sin + «ι (1). J

In this formula b = baseline, aO = amplitude, al = phase, and a2 = wavelength. The fit can be performed using the "Peakfit" software package øandel Scientific, Corte Madera, CA). Typically there will be ~80 to ~700 data points in a fit, depending upon the scan. Processing of digital images should be deliberately minimized. It should involve no more than background subtraction and contrast enhancement. The measured periodicities of chromatin fragment arrays are shown in Figure 7 for cells from normal control zebrafish embryos, and in Figure 8B for cells from camphothecin treated embryos. Example 11

Generation of Chromatin Beads Which Detach from an Underlying Matrix, and Which Diffuse in the Surrounding Medium by Brownian Motion: Measurements of Brownian Motion of Beads Zebrafish embryo animal cell caps are prepared in the usual fashion (of

Example 1), and are treated with the caspase-3 inhibitor Ac-DEVD-CHO during the lysis and spreading process. This treatment causes a significant number of the DNA-containing beads (which are produced in the spontaneous fragmentation of spreads) to detach from an underlying nuclear "substrate" or "matrix". Detached beads are also produced in untreated spreads although perhaps at a lower frequency. The detached beads are found to diffuse freely through the surrounding medium. Under -12,000 X total magnification, using a low- light (0.07 lux) CCD camera (Cohu, San Diego, CA), the inventors have been able to track the Brownian motion of these beads in real time. For example, 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). Assuming independent movement along the x and y dimensions, the diffusion path of an individual bead can be analyzed with the following formula:

< x ) + < y²> = 4D_T(0bsf (2)

where <x²> , <y²> = average of the square of the displacement of the bead from the origin along the x, y coordinate respectively, D_{τ 0\},_s = observed translational diffusion coefficient at temperature T, and t = time. (See Berg, H.C "Random Walks in Biology." Princeton University Press, 1983)

Stokes's law can then used to calculate a theoretical diffusion coefficient D_{τ caic} with the formula:

where k = Boltzmann's constant, T = absolute temperature, η_{T w} = viscosity of water at temperature T, and R = bead radius. A discrepancy between values of D_{τ obs} and D_{τ ∞fc} can be ascribed to a difference between the viscosity of the solution under the coverslip, and the viscosity of pure water. Figure 6A-F is a series of graphs showing the physical properties of beads produced by spontaneous fragmentation of the chromatin spreads. (A-distribution of bead volumes, from one nucleus. B-Gaussian distribution of random-walk step sizes, for bead of radius R=0.8μm, measured at 15 sec. intervals at T=22-23°C C-random walk path of single bead of radius R=0.8μm, measured at 15 sec intervals. D - random walk path of single bead of radius R= 1.8μm, measured at 30 sec intervals. E-displacement (μm) as function of (t)^ for bead of radius R=0.8μm. F-displacement (μm) as function of (t)^ for bead of radius R=1.8μm.) Example 12 Demonstration of Solid-Liquid Crystal Transition, for Individual Chromatin Beads Spreads are prepared and incubated at 2-10°C for 72 hours. This results in spontaneous fragmentation of spreads, to produce periodically-spaced beads. At this temperature, the beads appear to be solid in phase. Next, spreads are removed from 2-10°C, and transferred to 15-37°C This rapidly produces a change in the phase of the beads, such that they become liquid-crystalline. Under epifluorescence microscopy, using a 40-100X objective and 10-20X secondary magnification, the fusion of adjacent liquid-crystalline beads can be directly observed, and even tracked in real time. Figure 12A-C shows the liquid-crystalline behaviour of the beads. Example 13

Creation of Non-Homogeneous Surfaces. Containing Wells or Obstacles, and Spreading of

Chromatin on These Surfaces, for Specialized Purposes

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. The colloidal particles do not adhere to the glass, and therefore can be brushed, washed, or sand-blasted away, to produce wells. The inventors have determined that two size-classes of holes are evident, with diameters of ~10μm and 50±20μm. (Examination under bright field microscopy at total magnification = 50X). We propose that 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. We further propose that it might be possible to trap a chromatin bead within one of the larger holes, and then use this as an in situ PCR chamber.

B. Vitrification. 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 ~5μm width, and 5-20 μm 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. Example 14

Conditions for Physical Removal of Chromosomes. 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

X secondary magnification. This implies that a 40X (NA 0.75) microscope objective can be used, in conjunction with an inverted microscope and a micromanipulated micropipette to remove the DNA-containing fragments, for the purpose of obtaining order pools of genomic clones from discrete chromosomal regions. Using the inverted microscope setup which is described above, it is possible to directly visualize the chromosomes and chromatin fragments during the microdissection procedure. Alternatively, it would be possible to employ a regular (non-inverted) microscope, with a 32-40X long working distance objective.

In this case, the working distance of the objective must be sufficient to allow manipulation of a micropipette, in the space between the objective and the sample. In this case, because of the lower NA of the long-working distance objective, it is necessary to employ electronic image detection and enhancement, to allow viewing in real time. A cooled CCD camera with >5megaHz transfer rate can be used. Alternatively, an image-intensifying camera, with higher gain but somewhat lower resolution, can be employed instead. Example 15

Rapid, one-pass sampled-sequencing of a particular genomic subregion.

(i) Using the method of the present invention, 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. (ii) 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.

(iii) The bead is transferred to a reaction vessel, and subjected to PCR-based amplification, using random primers. Then, the PCR products are cloned. (See Wei, J. et al., 1994).

(iv) Selected clones are subjected to rapid, one-pass automated fluorescent sequencing from both ends. (Smith, M.W. et al., 1994.)

(v) The sequence determinations are then compared to the GenBank and dbEST sequence databases, to search for matches to known genes.

Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims.

All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Below full citations for the references referred to in the specification are provided.

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Claims

WE CLAIM:

1. A method for preparing chromatin from cells 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; and

(c) allowing a transverse fluid flow to occur to release the chromatin which attaches to one or both surfaces.

2. A method according to claim 1 wherein said cells are in an unfixed, live state of the time at lysis.

3. A method according to claim 1 wherein said cells are obtained from a living embryo of a multicellular eukaryotic organism.

4. A method according to claim 1 wherein the surfaces are optically transparent, to allow microscopic examination of material between them.

5. A method according to claim 1 wherein at least one of said surfaces has non- covalent binding affinity for DNA and chromatin.

6. A method according to claim 5 wherein said non-covalent binding affinity is introduced by means of a chemical coating on at least one surface.

7. A method according to claim 6 wherein said chemical coating is selected from the group consisting of poly(L-lysine), polyacrylamide, and N-substituted polyacrylamide.

8. A method according to claim 1 wherein at least one of said surfaces has covalent binding affinity for DNA and chromatin.

9. A method according to claim 8 wherein said covalent binding affinity is introduced by means of a chemical coating on at least one surface, combined with addition of exogenous, reactive agent.

10. A method according to claim 9 wherein said chemical coating is selected from the group consisting of poly(L-lysine) and N-substituted polyacrylamide and said exogenous reactive agent is selected from the group consisting of formaldehyde, glutaraldehyde, acetaldehyde, and UV-light.

11. 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) incubating the chromatin for a period of time sufficient for the spontaneous cleavage of the DNA within the chromatin.

12. A method according to claim 11 wherein said cells are in an unfixed, living state at the time of lysis.

13. A method according to claim 11 wherein said cells are obtained from a living embryo of a multicellular eukaryotic organism.

14. A method according to claim 11 wherein, in step (d) the spontaneous cleavage of chromatin produces ordered arrays of chromatin fragments or DNA-containing beads.

15. 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.

16. A method according to claim 15 wherein the agent that cleaves the chromatin is a deoxyribonuclease enzyme.

17. A method according to claim 15 wherein, in step (d) the cleavage of chromatin produces ordered arrays of chromatin fragments or DNA-containing beads.

18. 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.

19. A method according to claim 18 wherein the temperature in step (f) is from about 15°C to about 37°C.

20. A method according to any one of claims 1 to 19 wherein the temperature in steps (a) to (c) is from about 2°C to about 10°C

21. A use of the chromatin prepared according to claim 1 as a substrate for in situ hybridization of probes which have binding affinity for DNA or protein targets within the chromatin.

22. A use of the chromatin according to claim 11 as a substrate for in situ hybridization of probes which have binding affinity for DNA or protein targets within said chromatin.

23. A use of the chromatin prepared according to the method of claim 1 to evaluate weak points in DNA, as revealed by separation of normally connected segments of chromatin, during the spreading process.

24. A use of the fragmented chromatin prepared according to claim 11 to investigate normal or aberrant apoptosis in cells.

25. A use of the fragmented chromatin prepared according to claim 11 to detect functional aberrations in a biochemical pathway which controls apoptosis.

26. A use of the fragmented chromatin prepared according to claim 1 as a substrate for chromatin-microdissection procedures.

27. A use of the fragmented chromatin prepared according to claim 11 or 15 as a substrate for chromatin-microdissection procedures.

28. A use of the fragmented chromatin prepared according to claim 11 or 15 to generate material for microtransport procedures.

29. A use of the fragmented chromatin prepared according to claim 28, to generate material for electrophoresis.

30. A use of the fragmented chromatin according to claim 28, wherein chromatin spreads are prepared between surfaces which present barriers to molecular transport procedures.

31. A use of the fragmented chromatin according to claim 29, wherein chromatin spreads are prepared between surfaces which present barriers to electrophoresis.