WO1993017128A1 - Methods for detection of chromosomal sturcture and rearrangements - Google Patents

Abstract

Methods and reagents for the in situ detection of chromosome structure or a region of a chromosome involved in rearrangements are disclosed. These reagents include a multiplicity of labeled probe DNA sequences that are complementary to different portions of the chromosome or chromosome region to be detected, and label specific antibodies conjugated to interdependent signal producing moieties. Selected pairs of the probes are contacted under hybridizing conditions with the chromosome or chromosome region of interest. Subsequently, said label specific antibodies are attached to the labels, resulting in the coupling of said moieties chemical reactions upon the addition of substrates. Consequently, a signal is produced at the chromosome region of interest that can be detected by optical means.

Description

METHODS FOR DETECTION OF CHROMOSOMAL STRUCTURE AND

REARRANGEMENTS

This invention relates to methods for detecting a site characterized by a genetically significant rearrangement event in targeted chromosomal DNA sequences which may occur at any location in any chromosome. This invention further relates to methods comprising steps of applying first and second labelled probes to a target nucleic acid at regions adjacent to said site, wherein the probes comprise DNA sequences which are complementary to the chromosomal DNA sequences of interest. One key element of this invention is that the labelled probe DNAs are then specifically associated with first and second interdependent signal producing moieties capable of interaction by the diffusion of a chemical substance to produce a detectable signal. With the addition of reagent , the first and second moieties are induced to produce a signal at the site of a genetically significant event, and the presence or absence of the signal is then optically detected.

This invention also relates to methods for revealing pre-existing fluorescent labels, both for revival of faded labels and confirmation of previous results.

Background Of The^' Invention

Chromosome structure is intimately related to the manner and_. mechanics of gene expression in normal cell function. Just as importantly, conservation of chromosome structure during cell division is necessary for transmission of genetic information from cell to cell, and from generation to generation. Often however, chromosome structure is changed and may forecast problems in gene function.

Alterations in chromosome structure often coincide with, and may be the cause of many inborn genetic disorders and degenerative diseases, including certain cancers. Such alterations may take the form of additional or absent whole chromosomes, or additional or absent portions of chromosomes. Chromosomes may also be rearranged, as by a translocation, so that different chromosomal regions come to be linked to each other. A host of other genetic defects, including inversions, amplifications, and outright deletions, can occur alone or in combination with the above named defects.

Some gross chromosomal alterations are detectable as diseases. Alterations such as additional or absent chromosomes may lead to, for example, Down syndrome (extra chromosome 21 matter), Turner syndrome (deleted X chromosome in females) or Klinefelter syndrome (XXY chromosomes). Alterations involving parts of chromosomes can produce, for example, chronic myeiogenous leukemia (C L) and acute lymphocytic leukemia (ALL), both thought to occur in the presence of the so-called Philadelphia chromosome, which involves a translocation between chromosome 9 and chromosome 22.

Karyotype analysis is currently used in diagnosis of the aforementioned maladies. A karyotype is essentially a tally of the number and characteristics of an individual's chromosomes. Conventional karyotype analysis is done by staining and visualizing metaphase chromosomes and the characteristic patterns (called bands) produced. See, for example, ACT Cytgenetics Laboratory Manual, 2nd Edition, at page 222, Margaret J. Barch. Ed.. (1991 ) Raven Press Ltd., New York, New York.

Such "banding analysis^* is meticulous and time consuming, owing to the difficulties involved in obtaining good "metaphase spreads" of chromosomes from cultured cβlls-a problem that is extremely difficult to overcome when working with intransigent cell types as are present in certain tumors. The band staining patterns, especially on abnormal chromosomes, may be difficult to classify owing to rearrangements. The banding technique is indeed limited when seen in light of other disadvantages, such as; (a) requirements for highly trained analysts that perform labor intensive, time consuming work; and (b) lack of resolution for altered chromosomal regions of less than 3-15 megabases, depending on the particular defect [see Laπdegren et al., Science.

242:229 (1988)]. The present invention provides methods for overcoming such limitations.

The aforementioned staining and banding techniques have recently been aided by the introduction of automated karyotyping systems, which allow the burden of labor, but not vigilance, of the trained analyst to be eased. Such systems operate by identifying and organizing chromosomes based on the appearance of the chromosomes in the field

• of view of a light microscope. Because the quality of staining and banding of chromosomes is inconsistent, a trained cytogeneticist must review ail results so obtained. In their current state of art, such systems are expensive, and still require highly trained analysts for operation.

Accordingly, there is a need for a less complex and more economical approach to such analysis. The present invention requires a simple light microscope for it's immediate application and, in addition, can readily be adapted to automation already available. The degree of training and judgement required of the analyst is much reduced as well.

With the improved approaches to karyotypiπg analysis, more recent advances have come in the form of rβcombinant DNA methodology combined with in-situ hybridization techniques. Using the in-situ method, discrete nucleic acid probes obtained from purified DNA "libraries" have been used to map specified locations on chromosomes.

The process of hybridization occurs when DNA in either a fixed chromosome or a free probe is "denatured", or unravelled from its normal duplex, or double stranded, form. The resulting single stranded nucleic acid probe sequences will only "renature^* with their precise complement in the chromosome, thus binding to a specific location. Normally, such a probe is labelled with radioactive isotopes and locates to the chromosome, where it may be visualized by autoradiographic techniques. Alternatively, when such a probe is labelled with a hapten, or antigen, it may be localized by the use of fluorescent stains linked to antibodies and seen in a fluorescence capable microscope. This latter method is referred to as Fluorescence In-Situ Hybridization (hereinafter "FISH") See, for example. Gray et. al. EPO publication no. 0 430 402 A2.

Both of the above techniques have the advantage of sequence specificity over chemical staining. This means that knowledge of the DNA sequence corresponding to a specific whole or partial chromosome, or a specific gene on a chromosome, can potentially lead to high resolution chromosomal analysis. The potential dangers of using hazardous radioactive materials obviates the use of radiolabelled probes in a diagnostic clinical setting, where thousands of samples are handled. This leaves sequence specified staining, for which we have mentioned a number of advantages, e.g. independence from banding patterns for identification and less stringent requirements for analyst training. Still, the important disadvantages that remain are; (1), the inability to distinguish rearrangements involving smalt regions of chromosomal material, as in cytochemical banding; (2), in the case of fluorescence labelled probes for FISH, the requirement for expensive fluorescence optics on the microscope, and; (3) the inherent difficulty of using certain high-sensitivity stains without obfuscating chromosome morphology necessary for karyotypic analysis. Accordingly, there is a need to deal with such. shortcomings before the in-situ methodology can mature into a routine, but extremely valuable clinical tool. The present invention has no such limitations, as it is an important and novel approach to the specific and precise labelling of such chromosomal rearrangements, and in addition requires a relatively simple phase optics equipped microscope.

Methods of using DNA probes in the analysis of DNA junctions resulting from translocations. inversions, etc. are known in the art. All but a few utilize sequence specific probes that require DNA sequence information for utility, and are therefore extremely limited in their applications. For examples, see the following references:

Carr, EPO 0 246 864. discloses a method for using DNA partial hybrid probes to locate complementary target sequences of interest.to form "split probes" that can be linked together to make a detectable signal in the form of a double stranded DNA with high thermal stability, or other distinguishing physical character. Again, this method requires that precise information on the region of interest be in hand before application can occur.

Wθismann, U.S. Patent 4.710.465, describes the construction of junction-fragment DNA probes that may span 20-2000 kilobases, for use in the localization of genes involved in inheritable disorders. As before, this method requires preexisting information on the gene region of interest before application can occur, and consequently has little use for the analysis of chromosome structure.

Stephenson, U.S. Patent 4,681.840, discloses a discrete DNA probe specific for human chromosome 22, for use in examining translocations in the so-called Philadelphia chromosome (Ph'-between chromosomes 9 and 22). The probe DNA is used in a Southern blotting 8

diagnostic application but is not usable for in-situ chromosome analysis owing to its low complexity.

Methods for combining two different enzyme activities to produce a detectable signal are long known in the art.

Ullman, EPO 0 230 768, discloses methods of separating substances from a liquid medium, in which the presence of desired aggregates is determined by the formation of so-called complementary specific binding pairs, or "sbp's", which have been conjugated to selected enzymes. The sbp's are detected by a signal producing system comprising the combination of enzymes linked to sbp's that interact to produce a measurable signal, or product. No application of interacting enzyme pairs for use with DNA probes to chromosome structure is mentioned, although a DNA-DNA or DNA-RNA hybrid is mentioned as a possible sbp.

Similarly, Litman. U.S. Patent 4,275,149, describes the use of enzyme-particle and enzyme-sbp conjugates in an antigen-antibody context, where the enzymes are chosen to form a signal producing system. In the Litman patent, assay methods are described that depend on the presence of an analyte as part of the detection and signal producing scheme, which also utilizes a coupled enzyme system.

However, there is nothing suggested or taught about the use of the sbp's in combination with labelled DNA probes.

Methods for applying DNA probes in conjunction with immunological based signal targeting techniques are known in the art. Gray, et.al., EPO Application No.90308718.7. discloses methods and compositions for chromosome specific staining using "direct" fluorescence labelled probes comprising high complexity DNA sequences from individual human chromosomes. The specific applications call for the detection of chromosomal rearrangement by the microscopic detection of two different (color) fluorescence signals emanating from adjoining regions of FISH treated chromosomes, but the development of fluorescence does not depend on the presence of a rearrangement site.

Wiegant, et al, Nucleic Acids Res. 19, 3237 (1991), discloses the use of fluorescein-dUTP in a nick-translation format to produce fluorescein labelled human nucleic acid probes. The probes are used for ^• in-situ hybridization of human metaphase chromosomes, and also serve as targets for cytoimmunologicai enhancement via anti-fluorescein antibodies carrying yet more fluorescein labels. There is, however no teaching whatsoever regarding the enhancement of faded flouresceπt signals using visible dye production as taught in the present application.

While the fluorescent label techniques solve some of the problems of karyotypic analysis, and are improvements over existing banding methodology, they nonetheless fall short of providing a simple conditional test of a rearrangement and requires expensive fluorescence equipped microscopes. In addition, a clear disadvantage of using the fluorescent labels, as opposed to visible dye labels, is the inevitable fading of such fluorescent signals.

Methods for the combination of discrete DNA probes with immunological targeting and interactive enzymes to produce signals, is known in the art. Taub. U.S. Patent 4.820.630. discloses methods for the use of discrete DNA probes having associated labels that can interact enzymatically or physically to produce a signal. The discrete DNA probes are described as specifying a sequence to be analyzed for the presence or absence of a restriction site, or a "region of biological significance". However, the target sequences referred to in these examples are not chromosomes and the DNA probes are not of high complexity, as required for the analysis of vaguely defined genetically important regions. Further, the teaching of this patent specifies "one or two" discrete labelled DNA probes, and clearly could not operate where the "region of biological significance" was not already well characterized as by restriction mapping or sequence analysis.

Summary and Objects of the Invention

It is the general object of the present invention to provide methods for detecting normal and altered regions in chromosome structure using in-situ techniques in combination with reagents capable of localizing said regions by signal production.

It is an object of the present invention to provide a method for detecting alterations in chromosome structure using in-situ techniques in combination with reagents capable of localizing said alteration by direct visualization of said signal.

A more specific object of this invention is to provide a means of diagnosing chromosomal aberrations such as translocations wherein regions of different chromosomes become linked, sometimes causing or thought to cause disease

The objects of this invention can be attained by detecting a site characterized by a genetically significant rearrangement event in targeted chromosomal DNA sequences, which site may occur at any location in any chromosome, by applying the steps comprising:

(a) applying a first probe and a second probe to a target nucleic acid at a first and second region adjacent to said site, wherein said first probe has an attached first label, and comprises high to moderate complexity DNA sequences which are complementary to substantially all of the chromosomal DNA sequences of said first adjacent region and is able to attach to said first region of the target, and wherein said second probe has an attached second label, and comprises high to moderate complexity DNA sequences which are complementary to substantially all of the chromosomal DNA sequences of said second adjacent region and is able to attach to said second region,

(b) contacting the labelled product of step (a) with first and second interdependent signal producing moieties, said first interdependent signal producing moiety (ISPM) capable of attaching specifically to said first label by immunological means, and said second interdependent signal producing moiety (ISPM) capable of attaching specifically to said second label by immunological means, wherein said first moiety and said second moiety are capable of interaction by the diffusion of a chemical substance to produce a detectable signal,

(c) adding reagent comprising chemical substance capable of inducing said first and second moieties to produce a detectable signal at a site of a genetically significant event, and

(d) optically detecting the presence or absence of said signal.

This invention provides methods, reagents and compounds for in- situ detection of a chromosomal translocation. The reagents comprise unhybridized high or moderate complexity probe DNA sequences which

^■ are essentially complementary to most or all regions of the chromosome or chromosome region to be detected. Complexity of probe DNA refers to the number of bases in sequences that are not repeated. Such probe DNA's are named whole chromosome paints (or WCP's^tm Imagenetics-

PO Box 3011 , Naperviiie, Illinois 60566-7011 )and possess covalentfy bound multiple labels that can react specifically with immunological reagents. The term "whole chromosome paints" refers to a probe or probe composition, such as a probe composition of this invention, which is adapted to contact or hybridize a target which comprises one predetermined (i.e., preselected) chromosome of a multi-chromosomal genome. Typically, one WCP of this invention is combined with a second

WCP so as to make possible the indirect staining and subsequent detection of one or more predetermined chromosomal regions.

Probes

The labelled probe DNAs useful in this invention comprise two essential moieties, namely a polynucleotide portion and a chemically combined label portion- The polynucleotide portion of the probe DNAs can be in the form of plasmids. cosmids. phagemids, yeast artificial chromosomes (YACs) or other episomal DNA fo7ms, as well as DNA fragments of large or small size, that can adequately locate (i.e. hybridize) to specific chromosomal target sequences in sufficient quantity and juxtaposition to serve the purposes on this invention. Preferred probe DNAs are whole chromosome paints, comprising high to moderate complexity DNA sequence fragments which are complementary to the chromosomal DNA sequences of interest-

The sources of the DNA sequence used in the invention include but are not limited to DNA isolated from specific chromosomes, or libraries of such DNA, prepared by methods well known to those with skill in the art. The individual chromosomes from which DNA is isolated can be prepared by any of a number of standard methods, such as flow cytometry of microcell or somatic ceil hybrids, or by direct isolation from individual metaphase or interphase cells. Another source of such DNAs are libraries of specific chromosomal DNA, prepared by standard methods and available from traditional sources known to those in the art, such as the American Type Culture Collection (ATCC) or other repositories of human or other cloned genetic material. While a large number of chromosome libraries are available from the ATCC, representative libraries are:

ATCC NO,

57738 57753 57754

57716 57744 57717 57748 57751

57719 57718 57700 57745 57720

57746 57721 57701 57722 57755

57723 57702 57724 57705 57725

57736 57726 57704 57727 57736

57728 57705 57739 57706 57707

57729 57740 57737 57765 57730

57749 57758 57741 57759 57742

57710

57731 19 57766

19 57711

20 57732

20 57712 21 57743

21 57713

22 57733 22 57714 X 57750 X 57734

X- 57752

X 57747

Y 57735

Y 57715

The ATCC deposits are available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland. The invention contemplates that such DNA sequences may also be synthesized in - vitro by any of a number of enzymatic means known to those in the art. Also see an article entitled Human Chromosome-Specific DNA Libraries,

Biotechnology 4: 537 (1986). which describes the preparation of human chromosome libraries.

DNA used in the invention is isolated from these sources by methods which are well known to those skilled in the art. This DNA is then reduced to a heterogeneous mixture of variably sized fragments by any of a number of physical, chemical or enzymatic treatments, including but not limited to sonication, limited DNase I digestion, limited mung bean nuclease digestion, and sheanng of DNA through a narrow-gauge needle. The resulting mixture of DNA fragments are in a size range of 100-500 basepairs (bps) in length, although the preferred length of the average size of a fragment is about 300 bps. These procedures provide a large number of DNA sequences complementary to different portions of the chromosomes to be detected. In fact, thousands if not tens of thousands of DNA sequences complementary to different portions of the chromosome DNA are provided.

In order to label same, the DNA fragments are first derivatized by any of a number of chemical means known to those in the art to provide the DNA fragments with moieties capable of covalently bonding with appropriate labels, preferably by transamination of the carbon 4 (C-4) atom amino group of the nucleotide base cytosine. The derivatization results in the addition of a variety of reactive monoamme or diamine compounds at the C-4 position in this base, including but not limited to such compounds as hydrazine, alkylene diamines having 2 to 10 carbon atoms such as ethylenediamine, certain amino acids such as lysine or glutamine, and peptides, ether derivatives, or any of a number of other organic or inorganic linker molecules. Preferably, the DNA fragments have 5-25% of the cytosine residues contained therein transaminated.

The transaminated DNA sequences are covaiβntly linked to any of a number of labels comprising all compounds or entities which have a functional group capable of covalent bond formation with the transaminated DNA sequence, and are able to act as haptens in addition to other functionalities they may possess. By hapten is meant any chemical species that is able to be recognized and bound by an antibody, but which is not sufficient to illicit an immune response. Examples of such labels include but are not limited to, biotin (which may also interact with avidin and avidin-enzyme conjugates), phenyl and phenyl derivatives, caffeine and related compounds, fluoresciens, rhodamines and other fluorescent species, mercury or other metals, and any of a series of isoprenoids including carotenoids, sterols and steroids. Of particular interest are carboxytetramethylrhodamine (CTMR- obtainable from Molecular Probes, Inc.. Eugene, OR, catalog number

C1171 ), carboxyfluorescein (CFI-obtainable from Molecular Probes, Inc., Eugene, OR, catalog number C1311 ). theophylline, and dinitrophenyl (DNP), which are discussed within specific embodiments of this invention. Typically, the transaminated DNA sequences are reacted with an excess of functionalized label compounds and 60-80% of transaminated sites are labeled.

Alternatively, labels that are attached to previously hybridized DNA direct label probes clearly can also serve as the target of the ISPM (enzyme)-antibody conjugates in the dual enzyme system described above. A direct label probe is one that is designed to stain or otherwise distinguish the target DNA without subsequent labelling steps, such as with fluorescent labels. Many of the labels described in the prior art will operate well in such an application, since the only requirement would be that such labels are also haptens. In such an embodiment, a peroxidase type of ISPM in combination with added hydrogen peroxide and chromogenic substrate, can be used as a chromogenic system that targets fluorescent labels, specifically carboxytetramethylrhodamine (CTMR) and fluorescein and generally any of a series including rhodamiries, fluoresceins, umbelliferines, etc. The aforementioned enzyme-antibody conjugate system operates on "faded" fluorescent labels, as well as those freshly prepared. Thus applied, an anti- fluorescent label system allows recovery of the information for reanaiysis or confirmation from "old and faded" FISH treated metaphase spreads. The presently described method also provides a secondary analytical tool for FISH treated metaphase spreads.

In-situ Hybridization

This invention relates to the use of a multiplicity of different chromosome-specific probes having distinct antigenically active labels. Such specified labelled probes are hybridized to chromosomes or chromosomal regions, such as those involved in translocations and rearrangements. In particular, this invention relates to "in situ hybridization" of these chromosome specific probes to chromosomes from disrupted cells that have been prepared so as to leave the native chromosome structure essentially intact and to preserve the physical relationships between different chromosomes, different portions of the same chromosome or between chromosomes and other cellular structures. The term "m siiu hybridization" refers to the contacting or hybridization of a probe to a target in which hybrids are produced between a probe and a target. This term "in situ hybridization" is inclusive of denaturation and of a hybrid or probe detection procedure which is practiced after iasiiu hybridization of a probe to a target. In the present invention, a specimen can be adhered as a layer upon a slide surface. Targets for this hybridization include but are not limited to chromosomes or regions of chromosomes in normal, diseased or malignant human or other animal or plant cells, either interphase or at any stage of meiosis or mitosis, and either extracted or derived from living or postmortem tissues, organs or fluids; germinal ceils including sperm and egg cells, seeds, pollen, or zygotes, embryos, chorionic or amniotic cells, or cells from any other germinating body; cells grown in vitro, from either long-term or short-term culture, and either normal, immortalized or transformed; inter- or iπtraspecific hybrids of different types of cells or differentiation states of these cells; individual chromosomes or portions of chromosomes, or translocated, deleted or _,_ΛO 128

other damaged chromosomes, isolated by any of a number of means known to those with skill in the art, including libraries of such chromosomes cloned and propagated in prokaryotic or other cloning vectors, or amplified in vitro by means well known to those with skill; or any forensic material, including but not limited to semen, blood, hair or other samples.

Prior to hybridization, the labeled DNA sequences are preferably reacted with an excess of corresponding unlabeled DNA or reassociated fraction of unlabeled DNA for blocking non-specific hybridization. This blocking DNA is used at a concentration of 1-10 micrograms per 10 microliters of total genomic DNA, with a preferred range depending on the hybridized chromosome. The blocking DNA may be human placental DNA or Cot1 DNA (Cot . DNA supplied by Life Technologies, Gaithersburg, MD, Cat. # 5279SA). Briefly, Cot1 DNA is prepared by mechanically shearing total human genomic DNA to an average size of less than 400 base pairs. This material is denatured and then rehybridized for a period sufficient to render a large fraction of the highly repeated DNA sequences double-stranded. The mixture of double and single-stranded DNA species are treated with nuclease S1 , a nuclease that specifically degrades unhybridized single-stranded DNA to mono- and oligo-nucleotides. ^' Undigested, double stranded Cot1 DNA is recovered from this mixture.

Signal Production

The present invention addresses problems in the detection and identification of chromosomal regions which can be involved in translocations and rearrangements. The invention operates by producing signals resulting from the interaction of two interdependent signal producing moieties ("ISPM's").

The interdependent signal producing moieties (ISPM) can include catalysts, usually enzymes, and a plurality of substrates, and includes combinations of enzymes capable of interaction when the substrate of one enzyme is the product of the other enzyme. The final product of such interaction is the detectable signal, usually a visible dye or light signal, or a reactive chemical species able to interact with additional added components. A large number of such enzymes, substrates, and -, „-,,-„, 3/17128

combinations thereof are described by Littmann in U.S. Patent No. - 4,275,149, (1981).

In preferred embodiments, the ISPM's are two interdependent (i.e. coupled) enzymes. Combinations of enzymes that are of particular 5 interest include those which produce hydrogen peroxide and those which are able to use the hydrogen peroxide to oxidize a clear soluble substance to a detectable colored substance, such as a dye or other indicator. Examples of peroxide producers include, but are not limited to, glucose oxidase, gaiactose oxidase, aldehyde oxidase, xanthine 0 oxidase, monoamine oxidase, dihydroorotate dehydrogenase, and L- and D-amino acid oxidases. Examples of peroxide utilizers include horseradish peroxidase, microperoxidase, and cataiase.

Horseradish peroxidase is of particular interest because it can utilize a number of other compounds in addition to, or in conjunction with, 5 peroxide. In one embodiment, the enzyme alkaline phosphatase is able to convert 4-cJιloro-napthyl-1 -phosphate to 4-chloro-napthol. In conjunction with horseradish peroxidase and hydrogen peroxide, 4- chloro-napthol is converted to a an insoluble , dark purple dye, revealing the site of interest.

0 In yet another embodiment, horseradish peroxidase is paired with glucose oxidase. In the presence of O2 . glucose oxidase coverts β-D- glucose to D-giucono-<)-lactone and hydrogen peroxide. Horseradish peroxidase uses the hydrogen peroxide in conjunction with tetramethyibenzidine to produce a dark blue dye, and again reveals the

25 site of interest.

In embodiments of this invention, the aforementioned enzymes are conjugated to specific antibodies that correspond to chromosome specific probes. When said probes are in close proximity, and the proper reagents added, a visible signal is produced that is detectable using

30 simple light microscopes.

While enzymes are convenient and reliable catalytic ISPMs, a number of other biochemical and biophysical systems may be used in the practice of this invention. Substances formed by such systems are typically detectable by well known means, and include compounds such 28

as fluorophores, chromophores, chemiluminescent groups, odoriferous compounds¹, and others which have properties facilitating detection.

Embodiments of this invention utilize the compounds dinitrophenyi (DNP), theophylline, carboxytetramethylrhodamine (CTMR), or fluorescein for labelling of high complexity DNA probes. In combination with either a single or a coupled enzyme reaction comprising glucose oxidase and horseradish peroxidase, antibodies target the said enzyme activities to the hybridized probes with anti-theophylline, anti- dinitrophenol, anti-CTMR, or anti-fluorescein immunoglobuliπs (IgG's). Such reagent enzymes act either interdependently in the coupled system, or independently in the single system, to develop an easily visualized signal.

An important application of this invention is provision of a means to diagnose chromosomal aberrations such as translocations wherein regions of different chromosomes become linked, sometimes causing or thought to cause disease. Examples of such diseases are chronic myeiogenous leukemia (CML) and acute lymphocytic leukemia (ALL), both thought to occur in the presence of the so-called Philadelphia chromosome, which involves a translocation between chromosome 9 and chromosome 22. The breakpoints associated with this and other similar translocations are known to occur over a range of up to 150,000 bases. This fact obviates the use of so-called specific DNA probes for broad utility, since the fine structure of the breakpoint region would be required to target such discrete probes on their own.

The use of WCP's in combination with the ISPM's of this invention have no such disadvantage. To take advantage of the broad range of action of such high-complexity DNA probes in a diagnostic application, samples of chromosomes comprising the target DNA are prepared from cells of interest (for example, CML or ALL) and placed on a solid support such as a slide using well known in-situ fixation methods. Two of the above described labelled DNA probes, corresponding to different chromosomal sequences, are then hybridized to the fixed target DNA in the form of chromosomes, and sequentially treated with immunological reagents carrying interdependent (coupled) enzyme activities and reagents to catalyze a signal forming reaction, only in those chromosomal regions where a translocation has placed the enzymes - _{. .}

near to each other. Under a light microscope, a chromosome having a translocation appears to have a colored or stained segment attached to an unstained remainder segment, and the complementary pattern also appears on a corresponding chromosome.

DETAILED DESCRIPTION OF THF INVENTION

Efrpgπ^'πrøπtal Protocols

Utility of the coupled enzyme version of ISPM's was demonstrated by two different applications. The first shows the utility of the ISPM

1 0 methodology in producing detectable signal in an in-situ hybridization assay. A WCP probe for chromosome 1 was labelled with dinitrophenyl and hybridized to a normal lymphocyte metaphase spread. After hybridization, anti-dinitrophenyl goat IgG was applied to the hybridized labelled probe. Anti-goat IgG conjugated to horseradish peroxidase was 1 5 then reacted to the previously bound goat anti-dinitrophenyl antibody. The entire preparation was then perfused with a solution containing alkaline phosphatase. 4-chloro-napthol, and hydrogen peroxide. The reaction produced intense black staining over chromosome 1 , and very little background staining of other chromosomes.

20 In a second application, a translocation between chromosome 1 and chromosome 4 was detected using the present invention. In this specific example, the cell line sup B13 which contains the chromosome 1-4 translocation was the source of chromosomes for the preparation of metaphase spreads. Two different whole chromosome paint (WCP)

25 probes were used to detect the translocation. WCP for chromosome 1 was labelled with theophyliine. and WCP for chromosome 4 was labeled with dinitrophenyl (DNP). Horseradish peroxidase (HRP) was targeted to chromosome 1 via immunological means, and glucose oxidase (GOX) was similarly targeted to chromosome 4. In this example, the coupled

30 enzyme reaction was initiated by adding a glucose containing tetramethylbenzidine reagent. Glucose oxidase converts glucose to gluconate-delta-lactone and, more importantly, hydrogen peroxide is a byproduct of this reaction. Horseradish peroxidase converts a number of soluble, colorless products to insoluble dyes in the presence of hydrogen 35 peroxide. In the present example, the assay used tetramethylbenzidine. which is converted to a brilliant blue dye as the peroxidase substrate. As a result, two chromosomes were stained in all metaphases observed, in each case, only a portion of the chromosome was stained, the stained portion corresponding to that part of each chromosome derived from chromosome 1.

Elements of the present invention also provide a methodology that will amplify, retrieve or otherwise recover normally faded fluorescent in- situ hybridized ("FISH") labelled metaphase slides by restaining with the coupled enzyme chromogenic system of this invention. Such an application utilizes, for example, the fluorescent labels of previously hybridized probes as haptens for the attachment by immunological means of one or more of the ISPM's heretofore described. The method thus provided comprises the steps of contacting flourescent labels bound to previously hybridized probes with a signal producing moiety, wherein said moiety is directed to flourescent labelled chromosomes by immunological means comprising antigen/antibody or antibody/anti- antibody pairs, and reacting reagent comprising a first and second substance with said moeity. thereby converting a colorless soluble substrate to an insoluble detectable signal, said signal being in the range of visible light and detectable by optical means.

The following Examples are descriptions of the methods and reagents employed in the performance of the foregoing assays.

Example 1. Human Chromosome - Specific DNA Probes

Human chromosome-specific DNA probes were obtained as recombiπaπt phage libraries from Lawrence Livermore National Laboratories (LLNL) constructed as described in Van Diila, M.A. βt al. (Biotechnology 4: 537-552, 1986). These libraries were amplified by growth on an E. coli host strain. The amplified phage were purified, their DNA was extracted, and this DNA was digested with the restriction enzyme Hind III. Insert DNA was purified away from the lambda vector DNA and cloned into the Hind III site of the plasmid vector pBS (Strategene, La Jolla, CA). The resulting piasmids were transformed into an E. coli strain, DH5α (Bethesda Research Libraries, Gaithersburg, Maryland). The plasmid libraries used in this example are ATCC #'s 57738,

57753 an f 57754 (Chromosome 1 ); and ATCC numbers 57719, 57718,

57745, and 57720 (Chromosome 4). The libraries are stored as 1 ml aliquots of frozen cells. These vials have been used as the primary source for the production of seed stocks for fermentation.

Bacteria were grown by fermentation. The seed stock obtained from ATCC was cultured at 37°C for 24 hr. on 1.6% agar plates containing ampicilliπ (200 microgram/mi) and YT broth, which contains 8 grams per liter (g/l) of Bacto Tryptone (Difco), 5 g l of Bacto Yeast Extract (Difco), 15 g/l of Bacto Agar (Difco), and 5 g/l of sodium chloride. The cultured cells were harvested with 4 ml containing 16 g/l of Bacto Tryptone (Difco), 10 g/l of Bacto Yeast Extract (Difco) and 5 g/l of sodium chloride, and 4 ml of 20% glycerol was added to each harvest. The E. coli cell culture was quickly frozen in 0.5 ml aliquots by submerging the vials in liquid nitrogen and stored at -80°C until use.

The fermenter inoculum was prepared in 350 ml by cutturing the seed culture in a Casamino Acid medium which contains 13.2 g/l Na₂HPO4-7H₂0, 3.0 g/1 KH₂PO₄. 0.05 g/l NaCI, 1.0 g/l NH₄CI, 10.0 g/l Casamino Acids (Difco); 0.03 g/1 MgSO₄. 0.004 g l CaCI₂-2H₂0, 3.0 g/l glucose, 0.025 g/l Thiaminβ-HCI. 0-0054 g/1 FeCI₃, 0.0004 g/l ZnS0 , 0.0007 g/l CoCI₂, 0.0007 g/1 Na₂MoO₄. 0.0008 g/l CuSO₄, 0.0002 g/l H₂BO₃, and 0.0005 g/1 MnSO₄ in a 2 liter baffled shaker flash at pH 7 and 37°C. The 350 ml culture was used to inoculate 4.2 liters of fermentation media containing 1% glucose, 13.2 g/l Na₂HPO4-7H₂0, 3.0 g/l KH₂PO₄, 0.05 g/1 NaCI. 1.0 g l NH₄CI. 10.0 g/l Casamino Acids (Difco), 0.03 g l MgSO₄. 0.004 g/1 CaCI₂-2H₂0. 0.025 g/l Thiamiπe-HCI, 0.0054 g/l FeCI₃, 0.0004 g/l ZnSO₄, 0.0007 g/l CoCI₂, 0.0007 g/l Na₂MoO₄, 0.0008 g/1 CuSO₄, 0.0002 g/1 H₂BO₃, and 0.0005 g/l MnSO₄.

Bacterial cells were harvested employing a membrane cell- concentrator and a high speed centrifuge immediately after completion of the fermentation. The fermented cell broth was concentrated from 5 liter to approximately 800 ml employing a 0.45 micron (_m) membrane filter (2 square feet). The cell concentrate was then centrifuged at 7,000 x g for 10 minutes in a refrigerated centrifuge. The bacterial cell pellets are recovered after discarding the supernatant. 8

Plasmid DNA was extracted from bacterial cell pellets. The cells were thoroughly resuspended in 3 times the cell pellet mass (M) (in milliliters) of a solution containing 50mM glucose (fitter sterilized), 10 mM NaEDTA (pH 7.5-8.0), and 25mM Tris-HCI (pH 8.0). The cells were lysed with vigorous swirling after the addition of 6xM (in milliliters) in a solution containing 0.2 M NaOH, and 1% (w v) sodium dodecylsulfate (SDS). When the solution cleared, 4.5xM (in milliliters) of a solution containing 55.5 mi of glacial acetic acid and 147.5 grams of potassium acetate in a final volume of 500 ml was mixed thoroughly resulting in the production of a flocculent precipitate. The supernatant was separated from the flocculent precipitate and this supernatant centrifuged for 15 minutes at 7000 x g to remove residual precipitate.

Nucleic acid was precipitated from the supernatant with one volume of ethanol followed by centrifugation for 10 minutes at 7000 x g, and the nucleic acid pellets were resuspended in a total of 0.54xf "in milliliters). The nucleic acid was then extracted with 1/2 volu of neutralized phenol and 1/2 volume of chloroform and precipitated with two volumes of ethanol. The nucleic acid was resuspended in 0.3xM (in milliliters) of a solution of 50 mM Tris HCI (pH 7.0) and 100 mM sodium acetate. 0.77xM (in microliters) of 10 mg/ml RNase (heat treated) was then added and allowed to digest for 30 minutes at room temperature or overnight at 4°C. 0.615χM (in microliters) of a solution of Proteinase K (20mg/ml) was then added and incubated at 55°C for three hours. DNA was extracted with 1/2 volume of neutralized phenol and 1/2 volume of chloroform and precipitated with two volumes of ethanol.

DNA v .ia resuspended in 0.415xM (in milliliters) of water, and 0.05xM milliliters of 5 M NaCI and 0.155xM milliliters of 50% (w/v) polyethyleneglycol (PEG) (molecular weight 6000-8000) were added, incubated on ice water for one hour and precipitated by centrifugation for 15 minutes at 7,000 x g. The DNA was resuspended in 0.04xM milliliters of water and 1/10 volume of 3M sodium acetate and extracted with 1/2 volume of neutralized phenol and 1/2 volume of chloroform and precipitated with two volumes of ethanol. The purified DNA was resuspended in 0.0476xM milliliters of deionized H₂0. The DNA concentration was determined by fluorometry. 8

Finally, the purified DNA was disrupted into small fragments of approximately 300 base pairs by sonication using a Branson Sonifier 450 (Danbury, Connecticut). This size of fragments has been empirically determined to be the optimum for DNA probes used for in situ hybridization. Four milligrams of the purified plasmid DNA prepared above was resuspended in 2 mis of water and immersed in a dry ice/ethanol bath to prevent boiling during sonication. The microtip of the sonication device was- immersed in this solution until the tip was 2-5mm from the bottom of the tube. Sonication was carried out at an output power of 25-30 watts, discontinuously, with an 80% duty cyle (on 80% of time, off 20% of time), for a period of 5 minutes. Following sonication, the DNA was precipitated by the addition of 0.2 ml of 3 M sodium acetate (pH 5.5) and 4 ml of ethanol. The precipitate was recovered by centrifugation for 5 minutes at 8,000 x g and vacuum dried.

Example 2. Bisulfite Catalyzed Transamination of DNA

DNA obtained by the method of Example 1 was transaminated by the addition of ethylenediamine to the C4 carbon atom of the base cytosine. This reaction is catalyzed by sodium bisulfite. To prepare the bisulfite buffer, 1.7 ml of fuming HCI was slowly added to 1 ml deionized

H₂0 on ice. 1 ml fresh ethylenediamine (Sigma cat. #E-4379) was then slowly added on ice. After dissolution of the ethylenediamine, the solution was warmed to room temperature and 0.475 g sodium metabisuifite (Aldrich Cat. #25.555-6) was added. Fuming HCI was then slowly added to the bisulfite mixture until the pH reached 7.0. Deionized water was added to a final volume of 5.0 ml. To transaminate DNA, 1 milligram of sonicated DNA was resuspended in 0.3 ml. H₂0. The DNA was denatured by boiling at 100°C for 5 minutes then quickly chilled in an ice water bath. The transamination reaction was initiated by the addition of 0.3 mi of this DNA solution to 2.7 ml of bisulfite buffer, and the reaction was incubated at 37°C for 2 days. The DNA solution was desalted by routine dialysis against 5-10 miliimolar sodium borate (pH 8.0). After dialysis, 0.3 ml of 3 M sodium acetate (pH 5.5) was added to the diaiysate. The aminated DNA was precipitated with 2.5 volumes of ethanol and recovered after centrifugation at 8,000 x g for 10 minutes. The pellets were vacuum dried and rehydrated at a concentration of 3 mg/ml DNA. This solution was stored at -80°C until use.

Example 3: Preparation of Nitroohenvl Derivatives of Amino Acids

A solution of 20 mM ε-amino-π-caproic acid was prepared by adding 2.62 g of this compound to 20 ml water containing 40 mmol sodium bicarbonate. This solution was mixed with 20 ml of a 20 mM solution of Sanger's reagent (2,4 dinitro-fiuorobenzene) and allowed to stand at room temperature for 1 hour. The mixture was then gently heated, which caused the solution to turn yellow and a small amount of the dissolved sodium bicarbonate to precipitate. This precipitate was re- dissolved by the addition of a sufficient quantity of concentrated HCI and then left at 4°C to induce crystallization. The crystals were collected in vacua and washed with water to yield 4.2 g of yellow crystalline ε- dinitrophenylamino-t7-caproic acid (DNP-NCA).

DNP-NCA was activated by esterification to 3-sulfo-N- hydroxysuccinimide as follows. 0.594 g of DNP-NCA, 0.468 g dicyclohexylcarbodiimide and 0.434 g of 3-sulfo-N-hydroxysuccinimide were vigorously stirred in 7 ml dimethylformamide at room temperature overnight. This reaction was determined to have gone > 90% to completion by thin layer chromatoςraphy. The mixture was cooled to 0°C ' and stirred for an additional hour. The mixture was then filtered and the yellow solution evaporated to a thick yellow oil which did not crystallize. This oil was stirred with 50 ml ethanol to yield 0.996 g of a fine yellow powder which was collected by filtration and washed with ethanol. This compound is 6-N-(2.4 .1ιnitrophenylamino)caproic acid-O-(N- hydroxysucάnimidθ)-3-sulfoι.atθ (sodium salt) and will be referred to for the purposes of this invention as S-NHS-DNP.

Example 4: DNA Labeling: DNP-Deriviatived ε-amino-n-Caproic Acid

Chromosome-specific DNA of average length of about 300 bp prepared by the method of Example 1 was derivatized by bisulfite catalyzed transamination with ethylenediamine as described in Example 2. A solution of aminated DNA (100 μg total DNA) in a plastic 1.5 ml centrifuge tube was evaporated under reduced pressure. 0.5 ml of 0.2 M 3-[N-morpholino] propane sulfonic acid (MOPS) buffer and then 100 8

microliters of S-NHS-DNP (30mg/ml N,N-dimethyiformamide) was added to the residue and the mixture was incubated overnight at 25°C. DNP- Labeled DNA was precipitated by the addition of 60 μl of 3 M sodium acetate (pH 5.5) followed by 1.5 ml of ice cold ethanol and the mixture was incubated for at least 2 hours at -20°C. The solution was subjected to centrifugation for 10 minutes at 10,000 x g. The DNA pellet was washed twice with 0.6 mi of ice cold ethanol and then dissolved in 100 μl of sterile water. Two Sephadex G-50 Select D chromatography columns (5 Prime -> 3 Prime, Inc.) with a bed volume of 0.8 ml were prepared. 50 μl of the dissolved pellet was applied to each column and centrifuged for 4 minutes at 10,000 x g. 10 μl of the purified DNA was diluted with 490 μl of 20m NaOH and the optical density was determined at 260 nm to assess DNA concentration. The purified DNA was diluted with water to provide a working concentration of DNA of 100 micrograms per ml.

Example 5. Preparation of theophvlline-8-N-(5-hvdroxvpentvlamino -O- succinovl-O'-(N^,-f3-sulfosuccinimidvm ester

A solution of 5.16 grams of 8-bromotheophyliine and 5.15 grams of 5-amino-1 pentanol in 18 ml of p-xylene was refluxed for 18 hours. The reaction solution was allowed to cool and upon cooling to room temperature formed a solid/liquid mixture. The xylene was decanted and the solid was washed with pentane (3x40 miililiter). The resulting solid was stirred in 20 miililiter of water for 30 minutes and filtered to collect the solid material. The collected solid material was washed with water (3x20 miililiter), and dried to provide 3.73 grams of 8-(5-hydroxypentyiamino)- theophyliine. To a solution of 1.12 grams of 8-(5-hydroxypentylamino)- theophylline in 15 miililiter of anhydrous N.N-dimethylformamide (DMF) was added 0.516 gram of succinic anhydride and 0.2 gram of 4- dϊmethylamino pyridine (DMAP). The mixture was stirred with a magnetic stirrer overnight at room temperature under anhydrous conditions. The colorless solid that precipitated was collected by vacuum filtration, washed with dichloromethane and air dried to provide 1.20 gram of thθophylline-8-N-(5-hydroxypeπtylamino)-O-succiπic acid ester. This solid was recrystailized from a boiling 1 :1 propanol/water mixture and dried over calcium sulfate to provide a colorless solid. To a solution of 0.725 gram of theophyiiine-8-N-(5-hydroxypentylamino)-O-succinic acid ester in 15 ml of anhydrous dimethylformamide was added 0.414 gram of 3-sulfo-N-hydroxysuccinimide (Sulfo-NHS). A solution of 0.488 gram of dicyciohexyl-carbodiimide (DCC) in 2 miililiter of anhydrous DMF was added to the above solution. The resulting mixture was stirred with a magnetic stirrer at room temperature for 16 hours. The reaction mixture was cooled in an ice bath for 1 hour, then filtered under vacuum to remove dicyclohexylurea. The filtrate was evaporated in vacuo (2 torr, 30°C) to provide a viscous colorless oil. This oil was treated with 40 miililiter of anhydrous ethanol and a solid material precipitated. The precipitated material was collected by filtration and dried over anhydrous calcium sulfate to provide 0.450 gram of theophylline-8-N-(5- hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl)) ester, (NHS-theophylline).

ExamP'9 6 DNA Labeliπo - Theoohvlline

Chromosome-specific DNA probes to human chromosome 4 of average length of about 300 bp obtained by the procedure- of Example 1 were derivatized with the bisulfite catalyzed transamination with ethylenediamine as described in Example 2. Approximately 5% of the bases were aminated. A solution of aminated DNA (100 micrograms total DNA) in a plastic 1.5 miililiter centnfuge tube was evaporated under reduced pressure. 0.5. miililiter of 0.2 molar 3-[N-morpholino] propane sulfonic acid (MOPS) buffer and then 100 microliters of NHS- theophylline (26 miliigram/milliliter N.N-dimethylformamide) was added to the residue and the mixture was incubated overnight at 25°C. 50 microliters of 3 M sodium acetate (pH 5.5) were added followed by 1.5 miililiter of ice cold ethanol and the mixture was incubated for at least 2 hours at -20°C. The solution was subjected to centrifugation for 10 minutes at 10,000 x g. The pellet was washed twice with 0.6 miililiter of ice cold ethanol and then dissolved in 100 microliters of sterile water. Two Sephadex G-50 Select D chromatography columns (5 Prime -> 3 Prime, Inc.) with a bed volume of 0.8 miililiter were prepared. 50 microliters of the dissolved pellet was applied to each column and centrifuged for 4 minutes at 10,000 x g. 10 microliters of the purified DNA was diluted with 490 microliters of 20 miliimolar NaOH and the opf^-al density was determined at 260 nm to assess DNA concentration. Tne purified DNA was diluted with water to provide a working concentration of DNA of 100 micrograms per miililiter. Example 7. DNA Labeling: 5-fand-βVcarboxvtβtramβthvlrhodamιne. succinimidyl ester. fCTMR^

Chromosome-specific DNA probes to human chromosomes 1 and 4 of average length of about 300 bp obtained by the procedure of Example 1 were derivatϊzed by the bisulfite catalyzed transamination with ethylenediamine as described in Example 2. Approximately 5% of the bases were aminated. A solution of aminated DNA (50 micrograms total DNA) in a plastic 1.5 miililiter centrifuge tube was evaporated under reduced pressure. To this solution was added 377 microliter of 0.2 Molar 3-[N-morpholinoj propane sulfonic acid (MOPS) pH 7.4 buffer. Twenty- two and eight tenths microliters of CTMR (5-(aπd-6)- cartoxytetramethylrhodamiπe, succinimidyl ester, 50 miliimolar in N.N- dimethylformamide) was added to the transaminated DNA and the mixture was stirred overnight at -25°C (approximately 18 hours). The excess fluorophore was separated from the labeled DNA by ethanol precipitation. The precipitated DNA pellet was dissolved in sterile water, then passed over a Sephadex G-25 column that was 28 centimeters high with an internal diameter of 1 centimeter. The desired fraction (the column void volume) was eluted with water and dried to reduce the total volume. A second ethanol precipitation of the DNA completed the purification.

Example 8 Labeling of Transaminated Chromosome-Soecific DNA Probes with the Fluoropr.ore5-.and-6)-carboxyfluorescein. succinimidyl ester fCFI)

Transaminated DNA probes obtained by the method of Example 2 were conjugated with 5-(and-6)-carboxyfiuorescein, succinimidyl ester (CFI). Fifty micrograms of transaminated DNA were dried and then resuspended in 377 microliters of 200 mM MOPS, pH 7.4. Twenty-two and eight tenths microliters of 50 mM solution of 5-(and-6)- carboxyfluorescein, succinimidyl ester, (CFI) in N.N-dimethylformamide (a 150-fold molar excess) was added to the transaminated DNA. This reaction proceeded with stirring in darkness at room temperature overnight (approximately 18 hours). The excess fluorophore was separated from the labeled DNA first by an ethanol precipitation. The precipitated material was resuspended in water and passed over a Sephadex G-25 column that was 28 cm high with an internal diameter of 1 cm. The desired fraction (the column void volume) was eluted in water and dried to reduce the total volume. A second ethanol precipitation of the labeled DNA completed the purification. An absorbance spectrum showed that 1.6% of the bases were labeled.

Example 9 Preparation of Metaphase Spreads

SupB13 cells (CML line with multiple translocations case #10535) were made available by Michelle LeBeau, University of Chicago. Cells were reseeded into 25 cubic-centimeter flasks containing 80% RPMI 1640 media (Gibco catalog #320-1875), 20% fetal calf serum, 100 units Penicillin/Streptomycin, and 10 millimolar HEPES buffer. Cell growth was monitored by counting and cells were refed every 3 or 4 days. When cells had achieved optimal concentration, 0.2 ml 10 micrograms/milliiiter Colicimed (Gibco catalog#890-l 145-1) was added to each flask, and then held for 1 hour at 37C in a 5% CO2 incubator. Cells were harvested by centrifugation and resuspended in 5 milliliters 75 millimolar KCI, then held for 10 minutes at 37C. Cells were again harvested by centrifugation, and all but 0.2 miililiter of the KCL supernatant was removed. The ceils were dispersed, then fixed slowly in 10 milliliters 3/1 methanol/acetic acid, and held on ice for 15 minutes. Cells were harvested by centrifugation and placed in 5 milliliters fresh methanol/acetic acid, and held another 15 minutes on ice. Cells were harvested, resuspended in methanol acetic acid, and placed dropwise onto precieaned glass slides and dried.

Example 10 In Situ Hybridization Protocol

The coupled assay was tested by in situ hybridization using the standard technique. The target DNA was present in the sup B13 metaphases on a glass microscope slide. The slide was examined to locate areas containing nuclei and metaphase spreads. Outlines of the hybridization target area, and identification marks were made on the reverse side of the slide with a diamond scribe. Before hybridization, the target DNA on the slide was denatured by immersion for 2-10 minutes in a solution of 70% formamidβ/0.3 M NaCI/30 millimolar sodium citrate, pH 7.6-7.8 at 70° C. Following denaturation, the target DNA was placed in a 70% ethanol/water bath and agitated to remove the formamide solution. The wash step was repeated by passing the slide through 70%, 85% and 8

100% ethanol (ambient temperature, 2 minutes each). The hybridization mixture consisted of 50% formamide/0.3 molar NaCI/30 millimolar sodium citrate, pH 7.0. Human piacental DNA (2.25 microgram/10 microliter) was used as the blocking DNA. The theophylline labelled WCP 1 and the DNP labelled WCP 4 were each added to a concentration of 10 nanogram/microfiter. The total hybridization volume was 10 microliter. The hybridization mixture was denatured for 5 minutes at 70° C, then added to the slide over the area which contained the target DNA. A coverslip was placed on the hybridization mixture, and the edges were sealed with rubber cement. The slide was placed in a humidified chamber and hybridization proceeded overnight at 37° C. Following hybridization, the coverslip was removed and the slide was washed three times (5 minutes each) in 50% formamide/0.3 M NaCI/3 millimolar sodium citrate, pH 7.0 at 45° C. The slide was then washed 5 minutes in 0.3 M NaCI/3 millimolar sodium citrate and 5 minutes in 0.1 M sodium phosphate/0.1% NP40 (PN Buffer) each at 45° C. The slide was washed twice in PN buffer at room temperature, 2 minutes each wash. The slide was incubated for 20 minutes in anti-dinitrophenol (rabbit) diluted 1:250 in PNM buffer (PN buffer with 5% nonfat dry milk). The slide was washed 3 times in PN buffer at ambient temperature for 2 minutes each time. The slide was then incubated for 20 minutes in glucose oxidase-antϊ rabbit conjugate, and again washed 3 times in PN buffer. The slide was finally incubated for 20 minutes in horseradish peroxidase-anti theophylline conjugate and washed a final 3 times in PN buffer, with fresh buffer used for each washing step.

Example 11. Color Development and Result

A modification of the commercially available Vector TMB

(tetramethylbenzidine) peroxidase kit was used in the color development

Specifically, hydrogen peroxide was omitted from the substrate preparation and 100 microliters of 1 Molar glucose was substituted therein. The slide bearing the prepared, hybridized metaphase spread was incubated 1 hour with the chromogenic reagent, rinsed briefly under a gentle stream of distilled water, then air dried.

The TMB treated metaphase spread was counterstained for 5 minutes in freshly prepared, filtered Giemsa. The slide was rinsed under a gentle stream of distilled water. The slide was then dried with an air or nitrogen jet. The staineα metaphase spread was then examined in a microscope.

At least two chromosomes were labelled in all metaphases observed. In each case, only a portion of the chromosome was labeled, the labelled portion corresponding to that part of each chromosome derived from chromosome 1 . In some metaphases, a third chromosome was detectably labelled. In each case, this third chromosome was the normal copy of chromosome 1 .

Claims

That which is claimed is:

1. A method for detecting a site characterized by a genetically significant rearrangement event in targeted chromosomal DNA sequences, which site may occur at any location in any chromosome, comprising the steps of:

(a) applying a first probe and a second probe to a target nucleic acid at a first and second region adjacent to said site, wherein said first probe has an attached first label, and comprises high and moderate complexity DNA sequences which are complementary to substantially all of chromosomal DNA sequences of said first adjacent region and is able to attach to said first region of the target, and wherein said second probe has an attached second label, and comprises high and moderate complexity DNA sequences which are complementary to substantially all of the chromosomal DNA sequences of said second adjacent region and is able to attach to said second region,

(b) contacting the labelled product of step (a) with first and second interdependent signal producing moieties, said first interdependent signal producing moiety (ISPM) capable of attaching specifically to said first label by immunological means, and said second interdependent signal producing moiety (ISPM) capable of attaching specifically to said second label by immunological means, wherein said first moiety and said second moiety are capable of interaction by the diffusion of a chemical substance to produce a detectable signal,

(c) adding reagent comprising chemical substance capable of inducing said first and second moieties to produce a detectable signal at a site of a genetically significant event, and

(d) optically detecting the presence or absence of said signal.

2. A method of detecting genetically significant rearrangement events in a target DNA sequence, as recited in claim 1 , wherein said immunological attaching means includes at least one of the following combinations;

(1 ) first antibodies which are conjugated to first ISPMs, and capable of immunologϊcally attaching to first labels, and second antibodies which are conjugated to second ISPM's, and capable of immunoiogically attaching to second labels, or

(2) first antibodies which are conjugated to first ISPMs, and capable of immunoiogically attaching to first labels, second antibodies capable of immunoiogically attaching to second label, and third antibodies which are conjugated to second ISPMs, and capable of immunoiogically attaching to said second antibodies, or

(3) first antibodies capable of immunoiogically attaching to first label, second antibodies which are conjugated to second ISPMs, and capable of immunoiogically attaching to second labels, and third antibodies which are conjugated to first ISPM, and capable of attaching to first antibodies, such that said immunological means juxtapose said first and second ISPMs so as to produce a signal.

3. The method of claim 2 wherein said first label and said second label are non-identical and are each taken from one of three groups including; a) multiple xanthine or lower alkyl substituted xanthine derivatives; b) phenyl substituted with one to three nitro groups; and c) fluorescent compounds.

4. A method of detecting genetically significant rearrangement events in a target DNA sequence, as recited in claim 3, wherein said first and second ISPMs are a coupled enzyme system, comprising a first enzyme able to interact with a second enzyme by the diffusion of a chemical substance essential to producing the detectable signal.

5. The method of claim 1 wherein there are two probes, and each is high complexity whole chromosome paint (WCP) consisting essentially of chromosome specific labelled DNA fragments corresponding to locations over an entire individual chromosome.

6. The method of claim 1 wherein a first probe is a whole chromosome paint, and second said probe DNA. is either a yeast autonomous chromosome (YAC) clone or cosmid clone, said clones having insert sizes exceeding 50000 nucleotides.

7. A method of detecting and locating genetically significant rearrangement events in targeted DNA sequences, as recited in claim 1 , wherein said targeted DNA sequences are taken from a biological source of interest, and said DNA may be in the form of whole nuclei, chromosomes or fragments thereof, naked DNA or fragments thereof, where such DNA is either fixed to a slide so as to conserve the identifying morphology of distinct chromosomes or nuclei, or where such DNA is naked and bound to a solid substrate after a fractionation and separation means have been applied.

8. A method of enhancing or replacing signal from flouresenctly labelled in situ hybridized chromosomes, wherein said fluorescent label is faded or otherwise non-detectable, comprising the steps of;

(a) contacting fluorescent label portion of fluorescence labelled hybridized chromosomes with a signal producing moiety, wherein said moiety is directed to florescence labelled chromosomes by immunological means comprising antigen/antibody or anubody/antiantibody pairs; and

(b) reacting reagent comprising a first and. second substance with said moiety, thereby converting a colorless soluble substrate to an insoluble detectable signal, said signal being in the range of visible light and detectable by optical means.

9. A method of enhancing or replacing signal from fluorescently labelled in situ hybridized chromosomes, as recited in claim 1 , wherein said fluorescent label is carboxytetramethylrhodamine.

10. A method for detecting a junction site resulting from a translocation event in chromosomal DNA sequences, wherein targeted chromosomal DNA sequences are in the form of a metaphase spread, and wherein said site may occur at any location in any chromosome, comprising the steps of: (a) attaching a first probe and a second probe to a target chromosome at a first and second region at different sides of said site, wherein said first probe is a whole chromosome paint labelled with theophylline and comprises DNA sequences which are able to hybridize to said first region of the target, and wherein said second probe is a whole chromosome paint labelled with dinitrophenyl, and comprises DNA sequences which are able to hybridize to said second region,

(b) contacting said theophylline label with horseradish peroxidase anti- theophylline conjugate, and contacting said dinitrophenyl label, first with rabbit anti-dinitrophenyl and then with glucose oxidase anti-rabbit conjugate,

(c) adding reagent containing glucose and tetramethylbenzidine, capable of inducing said first and second moieties to produce a detectable signal at the junction site of a chromosomal translocation, and

(d) detecting the presence or absence of said signal in a light microscope.