CA2107880A1 - Methods for detection of chromosomal structure 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

~ WO93/17128 PCl/US93/01718 ~ ~;l 0788~

~ETHODS FOR DETECTION OF CHROMOSOMAL STRUCTU~E AND
REARRANGEMENTS

- This invention relates to methods for detecting a site characterized 5 by a genetically signiticant rearrangem~nt event in targeted chromosomal DNA sequences which may occur at any location in any chromosome. This invention turther relates to methods comprising steps of applying lirst and second labelled probes to a target nucleic acid at regions adjacent to said site, wherein the probes comprise DNA
l 0 sequences which are complementary to the chromosomal DNA
sequenc~s of interest. One key element of this invention is that the labelled probe DNAs are then specifically associated with first and second intardependent signal produang moieties capable of interaction by the diffusion of a chemical substance to produce a de~ectable signal.
1 5 With the addition cf reagent, the tirst and second moieties are induced to produce a signal at tha slte ol a gr~netlcally signilicant event, and the presenc0 or absence of the slgnal Is then optically detected.
;`This invention also relales to methods for revealing pre-existing iluorescent labels, both lor revlval ot laded labels and confirmation of 20 previous results.
~a~Lround O~ The Invenlion Chromosome structure is intimately rslatèd to the manner and ;Imechanics ot gene expression in normal coll fùnction. Just as importantly, conser~ration of chromosome stnucture during cell division is 2 5 necessary tor lransmission ot genetic information trom ce~l to cell. and from generation to generatlon. Otten how~ver, chromosome stnucture is changed and may torecast problems in gene function.
Alterations in chromosome stnucture ottsn coincide with, and may be the cause ot many inborn genetic disorders and degenerative 3 0 diseases, including certain cancers. Such alterations may take the form of additional or absent whole chromosomes, or additional or absent portions ot chromosomes. Chromosomes may also be rearranged, as by a translocation, so that difterent chromosomal regions come to be linked lo each olhcr. A host ot o~hcr genclic detecls, including invcrsions, '' ., .

wo g3,l,l28 2 1 0 7 8 8 1) PCr/US93/0171X

amplifications, and outright d~l0tions, can occur alon0 or in comDination with th3 a~ove named d~tects.
Som~ gross chrornosomal alterations ar~ dct~ctable as dis~as~s, Alt~rations such as additional or absent chromosom~s may lead to, tor 5 exampl~, Down syndrome (ex~ra chromosome 21 matter), Turner syndrome (deleted X chromosome in temales) or Klin~telter syndrorne (XXY chromosomes). Altcrations involving parts ot chromosomes can produc~, tor example, chronic myelogenous leuk~mia (CML) and acute Iymphocytic leukemia (ALL), both thought to occur in th~ presence ot the l O so-called Philadelphia chromosome, which involves a translocation between chromosome 9 and chromosome 22.
Karyotype analysis is currently used in diagnosis ot the atorementioned maladies. A karyotype is essentially a tally ot the number and charact~ristics ot an individual's chromosomes.
l 5 Conventional karyotype analysis is done by staining and visualizing metaphase chromosomes and the characteristic patterns (called bands) produced. See, tor exampls, ACT Cyt~enetics Laboratory Manual, 2nd Edition, at page 222, Margaret J. Barch, Ed., (1991) Raven Press Ltd., New York, New York.
2 0 . Such ~banding analysis- is mstlctJlous and time consuming, owing to the di~ficulties involved in obtalnlng good ~mGtaphase spreads~ ot chromosomes from cullured cells-a problem that is extremely ditticult to overcom~ when working wlth intransigent cell types as are present in certain ~umors. The band staining patterns, especially on abnormal 2 5 chromosomes, may be dl~ficult to classity owing to rearrangements. The banding technique is indeed limited when seen in light ot other disadvantages, such as; ta) requiremsnts tor highly trained analysts that per~orm labor intensive, time consuming work; and tb) lack of resolution lor altered chromosomal regions o~ less than 3-15 megabases, depending on the particular de~ect lsee Land~gren et al., Science.
.2,:229 (1988)]. The present invention provides methods tor overcoming such limitations.
The atorementioned staining and banding t~chniques have recently be~n aided by the introduction o~ automa~ed karyotyping 3 5 systems, which allow the burden ot ~abor, but not vigilance, o~ the trained analyst to be eased. Such systems operate by iden~:tying and organizing ' ~

~7880 chromosomes based on the appearance ot the chromosomss in the lield ot view ot~a light microscope. Because the quality of staining and banding of chromosomes is inco,lsislent, a trained cytogen~ticist must review all resu~s so obtained. 11~ ~heir current state ot an, such systems 5 are expensive, and still requir~ highly trained analysts tor operation.
Accordingly, there is a need tor a less complex and more economical approach to such analysis. The present invention requires a simple light microscope tor it's immediate application and, in addition, can readily be adapted to automation already available. The degree ot training and 10 judgement required ot the analyst is much reduced as well.
With the improved approaches to karyotyping analysis, more r~cent advances have come in the torm of racombinant DNA
methodology combined with in-sltu hybridization techniques. Using the in-situ method, discrete nucleic acid probes obtained trom puritied DNA
15 ~libraries~ have been used to map specified locations on chromosomes.
The process o~ hybridizatlon occurs when DNA in either a fixed chromosome or a lree probe is danatured-, or unravelled trom its normal duplex, or double stranded. torm. The resulting single stranded nùcleic acid probe sequences wlll only rsnatura with their precise complement 2 0 in the chromosome, thus blndlng to a speclfic location. Normally, such a probe is labelled Wl~ radloactlve Isotopes and locates to the chromosom~, where It may be visualized by autoradiographic techniques. Alternatively, when such a probe is labelled with a hapten, or antigen, it may be locall2ed by the use ol tluorescent stains linked to S antibodies and se~n in a tluorescenc~ capable microscope. This lattor method is reterred to as Fluorescencs In-Situ Hybridization (hereinatter ; ~FISH~) Sea, tor example. Gray el. al. EPO publication no. 0 430 402 A2.
Both ot ~he above techniques have the advantage ot sequence speciticity over chemical staining. This means Ihat knowledge ot ~he 3 0 DNA sequ~nco corr~sponding to a spscific whol0 or partial chromosom6, or a spocitic gene on a chromosome, can potentially lead to high resolution chromosomal analysis. The potential dangers ot using hazardous radioactive materials obviates the use ot radiolabelled probes in a diagnostic clinical setting, where thousands ot samples are handled.
3 5 This leaves sequence specified s~aining, tor which we have mentioned a number o~ advantages, e.g. independence trom banding patterns tor identitication and less stringent requirements tor analyst training.

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wo 93/1 71~8 2 1 0 7 8 8 0 PCI/US93/017 18 Still, the imponant disadvantages that remain are; (1), the inabili~y to distinguish rearrangements involving small regions ot chromosomal material, as in cytochemical banding; (2), in the case ot fluorescence Iabelled probes for FISH, the r~quirement for expensive fluorescence 5 optics on the microscope, and; (3) the inherent difficulty of using certain high-sensitivity stains wi~hout obtuscating chromosome morphology necassary for karyotypic analysis. Accordingly, there is a need to deal with such shortcomings before the in-situ methodology can mature into a routine, but extremaly valuable clinical tool. The present invention has 10 no such limitations, as it is an imponant and novel approach to the specific and prr~cise labelling ot such chromosomal rearrangemonts, and in addition requires a relatively simple phase optics equipped microscop~.
Methods ot using DNA probes in the analysis of DNA junctions 15 resulting from translocations, inversions, etc. are known in the art. All buta few util'ize sequence speciSic probes that require DNA sequence intormation for utility, and are therefore extremely limited in their applications. For axamples. see ~he ~ollowing reterences:
Carr, EPO 0 246 864, disclos~s a method tor using DNA partial 20 hybrid probes to locate complementary target sequences of interest.to form ~split probes~ Ihat can ~e linked together to make a détectable signal in the torm ot a double stranded DNA with high thermal stability, or other distinguishing physical character. Again, this method requires that precise information on the region ol interest be in hand betore 2 5 application can occur.
Weismann, U.S. Patenl 4,710,465, describes the construction ot junction-tragmcnt DNA probes that may span 20-~000 kilobases, tor use in tho locali2ation of genes involved in inheritable disorders. As betore, this method requires preexisting intormation on ths gene region ot 3 0 intsr~st bslors application can occur, and consequently has little use tor the analysis of chromosome stnucture.
Staphenson, U.S. Patent 4,681,840, discloses a discrete DNA
probe spscific tor human chromosome 22, for use in examining translocations in the so-called Philad~lphia chromosoms (Ph'-between 3 5 chromosomes 9 and 22). The probe DNA is used in a Southern bloning ~.

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, diagnostic application but is not usable tor in-situ chromosome analysis owing to its low complexity.
M~thods tor combining two differenl enzyme activities to produce a d~t0ctabla signal are long known in the art.
- 5 Ullman, EPO 0 230 768, discloses methods ot separating substances from a liquid medium, in which the presence of desired aggr~gates is determined by the formation of so-called complementary specific binding pairs, or ~sbp's~, which have been conjugated to sele~d enzymes. The sbp's are datected by a signal producing system l O comprising the combination of enzymes linked to sbp's that interact to produce a measurable signal. or product. No appllcation of interacting enzym0 pairs for use with DNA probes to chromosome structure is mentioned, although a DNA-DNA or DNA-RNA hybrid is mentioned as a ;l possible sbp.
- l S Similarly, Litman, U.S. Patent 4,275,149, describes the-use of enzyme-particle and anzyme-sbp conjugates in an antigen-antibody context, where the enzymes are chosen to form a signal producing system. In the Litman patent, assay melhods are described that depend on the presence of an analyte as pan oi the detection and signal 2 0 producing scheme, which also utili~es a coupled enzyme system.
However, there is nothing su~gested or taught a~out the use of the sbp's in combination with labelled DNA prob~s.
Msthods tor applying DNA probes in conjunction with immunological based signal ~arge~ing techniques are known in the art.
Gray, et.al., EF'O Applica~lon No.90308718.7, disclos~s methods and compositions tor chromosome specific staining using ~direct~
lluorescsnce labelled probes comprising high complexity DNA
sequenc~s trom individual human chromosomes. The specific applications call tor lhe detection ot chromosomal rearrangement by the 3 0 microscopic detection ot two ditferent (color) fluorescence signals emanating trom adjoining regions ol FISH treated chromosomes, but the development ot lluorescencs does not depend on the presence ot a rearrangement site.
Wiegant, et al, Nucleic Acids ~es. 19, 3237 (1991), discloses the 3 5 use ot fluorescein-dUTP in a nick-translation ~ormat to produce .. i, .
t WO 93/17128 PCr/US93/01718 - 2107~80 fluorescein lab~lled human nucleic acid probes. The probes are us0d tor in-situ hybrldization of human me~aphasa chromosomes, and also serv~
as targ~ts for cytoimmunological ~nhancement via anti-~luoresc~in antibodies carrying yet more fluor~sc~in lab~ls. Th~re is, howaver no 5 teaching whatsoever regarding the enhancement ot taded ~lourescnnt signals using visible dy~ production as taught in th~ pres~nt application.
While the fluorescent label techniques solve some ot the problems ot karyotypic analysis, and are improvements over existing banding methodology, they nonelheless tall short ot providing a simple 10 conditional test ot a rearrangement and requires expensive fluorescence equipped microscopes. In addition, a clear disadvantage of using the tluorescent labels, as opposed to visible dye labels, is the inevitable tading ot sùch fluorescent signals.
Methods tor th~ combination ot discrete DNA probes with 15 immunological targeting and interactive enzymes to produce signals, is known in the art. Taub, U.S. Palsnt 4,820,630, discloses methods for the use ot discrete DNA probes havlng associated labels that can interact enzymatically or physlcally to produc~ a signal. The discrate DNA
probes are described as specltylng a sequence to be analyzod for the 20 presence or absence ot a res~nctlon slte, or a ~region of biological signi~icance~. However, the targ0t sequences reterred to in these examples are not chromosomes and the D~A probes are not ot high complexity, as required tor the analysis of vaguely defin~d genetically important regions. Furthar, the teachlng ot this patent specifies ~one or 2 5 two~ discrr~te labelled DNA probes, and clearly could not oporats where the ~region ot biological slgnlficance was not already well characterized as by restriction mapping or sequenc~ analysis.

Summarv and Obj~ o~ the In~ention 3 0 It is the general object o~ ths present invention to provide methods ~or dstecting normal and altered regions in chromosome stnuCture using in-situ techniques in combination with reagents capable of localizing said regions by signal production.
It is an object ot Ihe present invention to provide a method tor 3 5 detecting altorations in chromosome stnucture using in-situ techniques in ; WO 93/17128 YCI/US93/01718 - ~107880 combination with r~ag~nts capabl~ of localizing said ~It~fation by direct visualization ot said signal.
A more specific object of this invention is to provide a means ot diagnosing chromosomal aberrations such as translocations wherein 5 regions ot different chromosomes become linked, sometimes causing or thought to cause disease .

The objects of this inYention can be attained by detecting a site charac:terized by a genetically si~nificant rearrangement event in targeted chromosomal DNA sequences, which site may occur at any 10 location in any chromosome, by applying the stQps 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 lirs~ label, and comprises high to moderate complexity DNA sequences which are c;omplementary to substantially all 15 of the chromosomal DNA sequences of said first adjacent region and is able to anach to said first region of the tarse~, and wh0rein said second probe has an attached second label, and comprises high to moderate complexity DNA s0quences which are compl~mentary to substantially all ot the chromosomal DNA se~uences of said second adjacent region and 2 0 is able to attach to said sscond region, (b) contacting the labelle~ product ol step (a) with lirst and second interdepondent signal producing moieties, said first interdrtpendent signal prodùcing moiety (ISPM) capable of attaching sptcifically to said tirst labrtl by immunologlcal means, and said second interdependent 2 5 si~nal producin~ moisty (ISPM) capabls o~ anachlng sprtcitically to said socond label by immunological means, wherein said first moiety and said socond moi~ty are capable of interaction by ~he diffusion of a chemical substanco to produce a detectable signal, (c) adding reagent comprisin~ chemical substance capable of 3 0 inducing said tirst and sscond moieties to proJuce a detectable signal at a sito ot a genetically significant event, and (d) optically detecting the presence or absence ot said signal.
~.
This invention provides methods, reagents and compounds lor in-situ detection ot a chrornosomal translocation. The reagents comprise ; 7 ~;,' ~;

WO 93fl7128 PCr/US93/0171~
21078~

unhybridized high or moderate complexity probe DNA sequences which are essentially complementary to most or all regions ot the chromosome or chromosome region to be d~tect~d. Complexity of probe DNA refers to the number ot bases in sequences that are not repeated, Such prob0 DNA s are named whole chrnrnosome paints (or WCP s'm Imagenetics PO Box 3û11, Naperville, Illinois 60566-7011)and possess covalently bound multiple labels that can react specitically with immunological raagents. The term ~whole chromosome paints~ reters to a probe or probe composition, such as a probe composition of this invention, which 10 is adapted to contact or hybridize a target which comprises one predet~rmined (i,e., preselected) chromosome ot 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 ot one or more predetermined chromosomal regions.
e~
:: The labelled probe DNAs use~ul in this invention comprise twoessential moieties, namely a polynucleotide portion and a chemically combined label portion, The polynucl~otide portion of the probe DNAs can be in the lorm o~ plasmlds, cosmlds, phagemids, yeast artificial 20 chromosomes (YACs) or other eplsomal DNA torms, as well as DNA
tragments of large or small size, that can adaquately locate (i.e.
hybridize) to specific chromosomal tar~st se~uences in sufficient quantity and ju~laposition to ssr~e ~he purposes on this invention.. Preterred probe DNAs are whole chromosome paints, comprising high to moderate 25 complexity DNA sequence tragments which are complementary to the chromosomal DNA sequences ot int~rsst.
The sources of the DNA sequence used in the invention include but are not limited to DNA isolated trom specitic chromosomes, or Iibraries of such DNA, prepared by methods well known to those with skill 3 0 in tho art, The indlvidual chromosomes trom which DNA is isolated can be prspar0d by any of a number oi standard methods, such as tlow cytomctry ot microcell or somatic cell hybrids, or by direct isolation from . individual motaphase or interphase cells. Another source ot such DNAs are librariss ot specific chromosomal DNA, prepared by standard 3 5 mcthods and available trom traditional sourt:es known to those in the art, such as the Am~rican Type Culture Collection (ATCC) or other repositories ot human or othe cloned genetic material. While a large ::
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PCr/US93/01718 ^wo 93/17128 !

~107~
number of chromosome libraries are available trorn th~ ATCC, reprasent~tive libraries are:
., , Humsn Chromo30me LlbrarY ATC~No.
: 1 57738 I 0 1 577~4 3 ~7717 4 . 57745 2 0 S ~7720 7 ~7722 . 8 57723 1 l 57704 3 5 12 5773~

17 577~9 . ~

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1 WO 93/1712~ PCr/US93/01~18 2~07~380 1 g 57711 ~7712 22 ~7733 ~; X 57747 The ATCC deposits are available from the Amorican Typs Culture Coll~ction, 12301 Parklawn Drive, Rockville, Maryland. The invention contemplates that such DNA sequences may-also be synthesized in -vitro by any of a number ot enzymatic means known to those in the art.
2 0 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 inventlon is isolated from these sources by methods which are well known to those skilled in the art. This DNA is 2 5 then reduced to a hetero~eneous mixture ot variably sized fragments by any of a number ot physical, chemlcal or enzymatic treatmenls, including but not limi1ed to sonicatlon, limited DNasa I digestion, limited mung bean nuclease digestion, and sheanng of DNA through a narrow-gauge needle. The resulting mixture ol ONA tragments are in a size range ot 3 0 100-500 basepairs (bps) in leng~h, althou~h the preterred length ot the average size ot a tragment is about 300 bps. These proceduros provide a largs number ot DNA sequenc~s complementary to different portions ot the chromosomos lo be detected. In tact. thousands it not tens ot thousands ot l:)NA sequences complementary to ditterent portions ot thq 3 5 chromosome DNA are provided.
In ordar to label same, the DNA tragments are tirst derivatized by any ot a number ot chemical means known lo those in the art to provide ths DNA tragments wilh moieties capable ot covalently bonding with appropriate labels, prsterably by transamination ot the carbon 4 (C-4) 4 0 a~om amino group ot the nucleotide base cytosine. Th~ derivatization r~sults in the addition ot a vanety o~ reactive monoamlne or diamine ." 10 ~;
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- ~:

~ WO 93/17128 21 0 r~ 8 ~313 pcr/lJs93/o17lx compounds at the C-4 position in this base, including but not limitea to such c~mpounds as hydrazine, alkylene diamines having 2 to 10 carDon . atoms such as ethylenediamine, certain amino acids such as Iysine or glutamine, and peptides. ether derivatives, or any of a nlJmber of oth~r organic or inorganic linker molecules. Preferably, the DNA tragm~nts hav~ S-2~% o~ the cytosine r~sidues contained therein transaminated.
The transaminated DNA sequences are covalently linked to any of a number of labels comprising all compounds or enlities which have a functional group capable of covalent bond formation with the transaminated DNA sequ~nce, and are able to act as haptens in addition to other tunctionalities they may possess. By hapten is msant any chemical species thal is able to be recognizad and bound by an antibody, but which is not sufficiant to illicit an immune response.
Examples of such labels include but ar~ not limited to, biotin (which may also interact with avidin and avidin-enzyme conjugates), phenyl and phenyl derivatives, calfeine and related compounds, fluoresciens, rhodamin~s and other fluorr~scent speci~s, mercury or other metals, and any of a ssries of isoprenoids including carotenoids, stsrols and steroids.
Ot particular interest are carboxyteSramethylrhodamine (CTMR-obtainable from Molecular Probes, Inc., Eugene, OR, catalog number C1171), carboxytluorescsln (CFI obtalnabla from Molecular Probes, Inc., Eugene, OR, catalog number C1311), theophylline, and dinitrophenyl (DNP), which are discusssd within specific embodiments o~ this invention. Typically, the transamlnated DNA sequences are reacted with 2 5 an excess ol tunclionalized lab~l compounds and 60-a0% of transaminated sites are labeled.
Alt~rnatively, labels that ars attached to previously hybridized DNA dir~ct lab~l probes clearly can also s~rvs as Ihe targot ot lhe ISPM
(en2ymo)-antibody conjugates in l~e dual enzyme system described ~, 3 0 abovo. A direct label probe is one that is designed to stain or otherwise distinguish tho target DNA without subsequent labelling steps, such as with lluor~scent labels. Many o~ the labels described in the prior art will - operato well in such an application, sinco the only requirement would be that such labels are also haptens. In such an embodiment, a peroxidase 3 S type ot ISPM in combination with added hydrogen peroxide and chromogenic substrate, can be used as a chromogenic system that targets ~luorescent labels, speci~ically carboxytetramethylrhodamme .

l l :

WO93/171~8 2107'~80 PCl/US93/O~

(CTMR) and tluorescein and generally any ot a series including rhodamiries, fluoresceins, umbelliterines, etc. The aforementione~
en~ym~-antibody conjugate syslem operates on ~faded~ tluorescent labcls, as w011 as those treshly prepared. Thus applied, an an~i-- 5 fluoresc~nt label system allows recovery ot the intormation for reanalysis or confirmalion Irom ~old and taded~ FISH treated metaphase spreads The prssently described method also provides a secondary analytical ~ool tor FISH treated metaphase spreads.
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This invention relatss 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 15 rearrangements. In partlcular, this invention relates to hybridiza~ion~ ot these chromosome specific probes.to chromosomes trom disrupted cells that ~lave ~een prepared so as to leave the native chromosome structure essentlally intact and to prsserve the physical relationships belween dlt~erenl chromosomas, different portions of the 2 O sarne chromosome or ~etw0en chromosomes and other cellular structures. The lerm ~ yondl2all0n^ relers lo the contacting or hybridization ot a pro~e lo a larget m which hybrids are produced belween a probo and a target. This terrn ~ Si1~hYbridization~ is inclusive ol denaturallon and ol a hybnd or probe detection procedure 2 5 which is practiced atter ~ ~ hybridization ot a probe to a tar~et. In the present invention, a speclmen can be adhered as a layer upon a slide surtacc. Targets tor this hybridization include but are not limiled to chromosomes or regions o~ chromosomes in normal, diseased or mall~nant human or olher animal or plant cells, eilher interphase or at 3 O any stags ot meiosis or mitosis, and eith~r extracted or defived from living or postmortem tissues, organs or tluids; germinal cells including sporm and egg cells, seeds, pollen, or zygotes, embryos, chorionic or amniotic cells, or cells trom any olhar germinatin~ body; cells grown in vitro, trom either long-term or short-term culture, and either normal, 3 5 immortalized or transtormed; inter- or intraspecitic hybrids ot ditterent Iypes ot cells or ditterentiation states o~ these cells; individual chromosomes or portions ot chromosomes, or translocated, deleted or .:`
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WO 93/17128 ~ 1 0 '7 ~ g O PCr/US93/0171X
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other damaged chromosomes, isolat~d by any of a number ot means - known to those with skill in the art, including libraries of such chromosomes cloned and propagatsd in prokaryotic or other cloning vectors, or amplified in vitro by means well known to those with skill; or 5 any torsnsic material, includin~ but not limited to semen, blood, hair or othcr samples.
Prior to hybridization, ~ha labeled DNA sequences are preferably react~d with an excess ot corrssponding unlabsled DNA or reassociatad traction oI unlabeled DNA tor blocking non-specific hybridization. This 10 blocking DNA is used at a conc~ntration ol 1-1û micrograms per 10 microliters of total genomic DNA, with a pret~rred range depending on the hybridized chromosom~. The blocking DNA may be human placental DNA or Cotl DNA (Cotl DNA supplied by Life Technologies, Gaithersburg, MD, Cat. # 5279SA). ~rietly, Cotl DNA is prepared by I S mechanically shearing total human g~nomic DNA to an average size ot less than 400 base pai!s. This material is denatured and then r3hybridized for a period sufficient to r~nder a large fraction ot the highly repeated DNA sequences double-stran~ad. Ths mixture of double and single-stranded DNA species are trsa~ed with nuclease S1, a nuclease 20 that specifically degrades unhybridi2sd singls-stranded Dl~iA to mono-and oligo-nucleotides. Undigested, double stranded Cotl DNA is recovered from this mixture.
Produ~
The pr~sent invention addresses problsms in the detection and 2 5 identification oI chromosomal regions which can be involved in translocatlons and rearrangements. Ths inven~ion operatss by producing si~nals rssultin~ trom the interac~ion oI Iwo interdependent signal producin~ moietles (~ISPM's-).
Tha intl3rdependent signal producing moieties (ISPM) can include 3 0 catalysts, usually onzymes, and a plurality ot substrates, and includes combinatlons ot enzymes capable of interaction when the substrate ot one en2yme is the product oI the other enzyme. lhe final product ot such ~; interaction is the detectable signal, usually a visible dye or light signal. or a reactiv~ chemical species able to interact with additional added 3 5 components. A large number ot such enzymes, substrates, and .~

WO 93/17128 P~r/us93/(~1r~
~107g~0 ,,, combinations th~reot are described by Littmann in U.S. Paterlt i'Jo.
4,275,149, (1981).
In preferred embodiments, the ISPM's ar~ two int~raepena~nt (i.c.
coupled) enzymes. Combinations ot enzymes that are ot particular 5 intarest 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 ot peroxide producers include, but are not limited to, glucose oxidase, galactose oxidase, aldehyde oxidase, xanthine 10 oxidase, monoamine oxidase, dihydrooro~at3 dehydroganase, and L-and D-amino acid oxidases. Examples ot peroxid~ utilizers include horseradish peroxidase, microperoxidase, and catalase.
Horseradish peroxidase is ot panicular interest because it can utilize a number ot other compounds in addition to, or in conjunc~ion with, 15 peroxide. In one embodiment, the enzyme alkaline phosphatase is able to convert 4-chloro-napthyl- 1 -phosphale to 4-chloro-napthol. in conjunction with horseradlsh peroxidase and hydrogen peroxide, 4-chloro-napthol is conven~d to a an insoluble, dark purpls dye, revealing the site ot interest 2 0 In yet another embodlment, horsaradish peroxidase is paired with glucose oxidase. In the presence 012 . 5~1ucose oxidase coverts ~-D-glucose to D-glucono-~lactone and hydrogen peroxide. Horseradish peroxidase uses the hydrogen peroxide in conjunction with t~tramethylbenzidine to produce a dark blue dye, and again reveals the 2 5 site ot interost.
In embodimonts ot this invention, ~he alorementioned enzymes are conJugaled to spocitic antibodies that correspond to chromosome specific pmbos. Whsn said probes are in close proximity, and the proper reagents added, a visibla signal is produced that is detectable using 3 0 simple light microscopes.
While enzymes are convenient and reliable catalytic ISPMs, a number ot other biochemical and biophysical systems may be used in tho pra~,tic~ of this invention. Substances tormed by such systems are typically detectable by well known means, and include compounds such .

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I'C'r/US93/0171X

~ ~ ~3 7~ J
as tluorophores, chromophores, chramiluminescent groups, odori~arous compounds~, and olh0rs which have propenies tacilitating detection.
Embodiments of this invention u~ilize the c~mpounds dinitropnenyl (DNP), theophylline, carboxytetrame~hylrhodamine (CTMR), or fluorescein tor labelling o~ high complexity DNA probes. In combination with either a single or a coupled enzyme reaction comprising glucose oxidase and horseradish peroxidase, anti~dies targ0t the said enzyme activities to the hybridized probes with anti-theophylline, anti-dinitrophenol, anti-CTMR, or an~ luorescein immunoglobulins (IgG~s).
1~ Such reagent enzymes act either interdependently in the coupled system, or independently in the single system, to develop an easily visualized signal.
An important applica~ion ot this invention is provision ot a means to diagnose chromosomal aberralions such as translocations wherein regions ot difterent chromosomas become linked, sometimes causir~g or thought to cause disease. Examplas ot such diseases are chronic '~ myelogenous leukemia (CML) and acute Iymphocytic laukemia (ALL), ~' both thought to occur in the presence ot the so-called Philadelphia chromosome, which'involves a translocation between chromosome 9 and chromosome 22. The ~reakpoints associated .with this and other '~' similar translocations ar0 known so oc~r over a range ot up to ,0,000 bases. This tact obviates tlle use ot so~alled specific DNA pro~es tor broad utility, since thr~ fine structure o~ the breakpoint region would be required to target such dlscrete probes on their own.
2 5 Tho use ot WCP's in combination with the ISPM's ot this inventionhave no such disadvantag~. To taka advantaga ot the broad range ot aaion o~ such high-complaxity DNA prob,es in a diagnostic application, samplas o~ chromosomes colnpnsin~ tho target DNA are prepared trom ;. cells ot intorest ~tor exampl~, CML or ALL) and placed on a solid support '''~ 3 0 such as a slid~ using well known iQ ~L~Y fixation methods. Two ot the above describ~d labelled DNA probos, corresponding to ditterent chromosomal sequencos, are then hybridized to lhe tixed targ~t DNA in ~h~ torm ot chromosomes, and sequentially treated with immunological r~agents carrying interdependent (couplsd) enzyme activities and 3 5 roagsnts to catalyze a signal lorming reaction, only in those chromosomal regions where a translocation has placed the enzymes I S

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WO 93/17128 2107 ~ 8 0 PCJ/US93/017,~

near lo each other. Under a light microscop~, a chromosome havlng a translocation appears to have a color~d or stainad segmenl attached to an unstained r0mainder segmant, and tha cornplem~ntary patt~rn also appears on a corr~sponding chromosom~.

DETAIL~ DESCRIPTION OF Tli~YENT!ON
ExDerir~ental Prot~s~ls Utility of the c~upled enzyme vsrsion of ISPM's was demonstrated by two dift~rent applications. The first shows ~he utility ot the ISPM
methodology in producing detectabl~ signal in an in-situ hybridization assay. A WCP probe tor chromosom~ 1 was labelled with dinitrophenyl and hybridized to a normal Iymphocyte mstaphase spread. Atter hybridization, anti-dinitrophenyl goat IgG was applied to the hybridized labelled probe. Anti-goat IgG conjugated to horseradish peroxidase was .; 15 then rsacted to the previously bound goat anti-dinitrophenyl antibody.
The entir~ preparation was th~n p~rtused with a solution containing alkaline phosphatase, 4-chloro-napthol, and hydrogen peroxide. The raaction produced intense bbck staining over chromosome 1, and very little background staining o~ other chromosomes.
In a second applica~ion, a translocation between chromosome 1 and chromosome 4 was detected using the present invention. In this specific example, the cell line sup ~13 which contains thc chromosome 1-4 translocation was the source ot chromosomes tor the preparation ot mstaphase spreads. Two ditterent whole chromosom~ paint (WCP) 2 5 probes w~re used to detect the translocation. WCP tor chromosome 1 was labelled with thoophylline, and WCP tor chromosome 4 was labeled with dinitrophenyl ~DNP). Horseradish peroxidasa (HRP) was targeted to chromosome 1 via immunological msans, and glucoss oxidase (GOX) was slmilarly targeted to chromosoma 4. In this example, the coupled 3 0 cnzym~ r~action was initiated by adding a glucose containing tetramethylben2idine roagont. (31ucose oxidase converts glucose to slucohate~delta 1actone and, more importantly, hydrogen peroxide is a byproduct ot this reaction. Horseradish peroxidase converts a number ot soluble, colorless products to insolubla dyss in the presence ot hydrogen 3 5 . peroxide. In the present example, the assay used tetramethylbenzidine.

wnich is c~nvened to a brilliant blua aye as th~ peroxidase substrat~, As a result, hYo chromosomes were stained in all m0laphascs observ0d In each case, only a ponion ot the chromosome was stained, th~ stain~d ponion corresponding to that part o~ each chromosome derivcd trom S chromosom0 1, El~ments o~ the present invention also provide a methodology that will amplity, retrieve or otherwise recover normally faded fluorescent in-situ hybridized (~FISH~) labelled metaphase slides by restaining with the coupled en2yme chromo~enic system o~ this invenlion. Such an 10 application utilizes, ~or example, the fluorescent labels ot previously hybridized probes as haptens for the attachment by immunological means of one or more o~ the ISPM's heretotore described. The method thus provided compris~s the steps of contacting flourescent labels bound to previously hybndized probes with a signal producing moiety, 1~ wherein said moiaty is directed to ~lourescent labelled chromosomes by immunological means comprising antigen/antibody or antibody/anti-antibody pairs, and reactlng reagent comprising a tirst and second substanc0 with said moelty, ~hereby converting a colorless soluble substrata to an insoluble ~a~ectable si~nal, said signal being in the range 2 0 ot visible light and deteaable by optlcal rneans.
The ~ollowing Examples ar~ descriptions of the methods and r~a~ents employed in the perlormance ot the toregoing assays~

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2 5 Human chromosome-speci~ic DNA probes were obtained as rocombinant phage libraries Irom Lawrence Livermore National Laboratories ~LLNL) constn cted as des~ribed in Van Dilla, M.A. et al.
(Liotechnology 4: 537-552, 1986). These libraries were amplilied by growth on an E. co/i host strain. Thc amplificd phage w0re purifi~d. their 30 DNA was ~xtractad, and this DNA was digestod with the restriction enzyme Hind 111. Insert DNA was puritied away ~rom the lambda vector DNA and cloned into the Hind 111 site ! the plasmid vector pBS
~Stratagr~no, La Jolla, CA). The resulting plasmids were transformed into an E. coli strain, DH5 (~ethesda Research Libraries, Gaithersburg, 3 5 Ma~land).
;

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wo 93/17~28 l~Cr/vS93/01~8 ~078~3 The plasmid libranes used in this ~xampl~ ar0 ATCC #'s 5773B, 57753 an~ 57754 (Chromosume 1); and ATCC numb~rs 57719, 57718, 5774S, and ~7720 (Chromosnme 4). The libran~s ar~ stored as 1 ml aliquots ol trozen cells. These vials have been used as the primary 5 source tor the production ot s~d stocks for f~rmentation.
~acteria were ~rown by termentation. The seed stock obtained trom ATCC was cuttured at 37C ~or 24 hr. on 1.6% agar plates ~ containing ampicillin (200 micrograrn/ml) and `fT broth, which contains 8 - grams per liter (9/1) ot Bacto Tryptone (Difco), 5 9/l of Bacto Yeast Extract 10 (Difco), 15 s/l o~ Bacto Agar (Difco), and 5 ~/1 of sodium chloride. The cultured cells were harvested with 4 ml containing 16 9/l of Bacto Tryptone (Difco), 10 g/l of Bacto Y~ast Extract (Ditco) and 5 9/l of sodium chloride, and 4 ml ot 20% glycerol was added to each harvest. The E.
coli cell culture was quickly trozen in 0.5 ml aliquots by submerging the 15 vials in liquid nitr.ogen and stored at -80C until use.
Ths termenter inoculum was prapared in 350 ml by cutturing the - seed culture in a Casamino Acid medium which contains 13.2 9/l Na2HP04-7H20, 3.0 gA KH2P04, 0.05 gJl NaCI, 1.0 9/l NH4CI, 10.0 gtl Casamino Acids (Ditco); û.03 ~ MgSO4, 0.004 9/l CaC12-2H20, 3.0 9/1 20 glucosc, 0.025 gJI Thiamlne-HCI, 0.0054 9/l FeC13, 0.0004 9/1 ZnSû4, - ~ 0.0007 9/1 CoC12, O.OOû7 ~ Na2MoO4, 0.0008 9/l CuSO4, 0.0002 9/l H2BO3, and 0.0005 gA MnSO4 m a 2 llter battled shakcr flash at pH 7 ;~ and 37C. The 350 ml culture was used to inoculate 4.2 liters oftermsntation media contalnmg 1% glucose, 13.2 9/l Na2HP04-7H20, 3.0 25 9/1 KH2P04, 0.05 9/l NaCI, 1.0 ~/1 NH4CI, 10.0 9/1 Casamino Acids (Ditco), 0.03 ~ SO4, 0.004 9/1 CaC12-2H20, 0.025 9/1 Thiamine-HCI, 0.0054 9/1 FeC13, 0.0004 9/l ZnSO4, 0.0007 9/1 CoC12, 0.0007 9/1 Na2MoO4, O.OOOB ~I CuSO4, 0.0002 ~I H2B03, and o.oaos 9/l MnS04.
Bacterial colls were harvestsd employing a msmbrane c911-3 0 conc~ntrator and a high speed csntritug~ immediately after compl~tion ot the fermentation. The termented cell bro~h was concentrated trom 5 liter to approximately 800 ml employing a 0.45 micron Lm) membrane tilter (2 square feel). The cell concentrate was then centritug~d at 7,000 x 9 tor 10 minutes in a re~rigerated centrifug~. The bacterial cell pellets are 3 S rscovered after discarding the supematant.

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Wo 93~17128 ~ r/us93/ol71x 0 7 g ~ ~
Plasmid DNA was exîracted trom bacteria~ ~11 p~llets. The cells were thoroughly resuspendad in 3 times the cell pellet mass (M) (in milliliters) ot a solution containing 50mM glucose (lilter sterilized), 10 mM
NaEDTA (pH 7.5-8.0), and 25mM Tris-HCI (pH ~.0). The cells wer~ Iysed 5 with vigorous swirling atter the addition ot 6xM (in millilit~rs) in a solution containing 0.2 M NaOH. and 1% (wlv) sodium dodecylsultate (SDS).
Wh~n the soiution cleared, 4.5xM (in milliliters) ot a solution containing 55.5 ml of glacial acetic acid and 147.5 grams of potassium acetate in a final volume of ~00 ml was mixed thoroughly resulting in the production 10 ot a tlocculent pracipitate. Tha supernatant was separated from ~he tloc~ulsnt precipitate and this supsrna~ant centrifuged for 15 minutes at 7000 x 9 to remove residual precipitate.
Nucleic acid was precipita~ed from tha supernatant with one volume ot ethanol tollowed by centritugation tor 10 minutes at 7000 x 9, 15 and the nucleic acid pellets w0re resuspended in a total ot 0.54xM (in milliliters). The nucleic acid was then extracted with 1/2 volume ot nqutraliz~d phenol and 112 ~olume o~ chlorotorm and precipitated with two volumes ot e~hanol. The nucleic acid was resuspended in 0.3xM (in . milliliters) ot a solution ol 50 mM Tris HCI (pH 7.0) and 100 mM sodium 20 acetate. 0.77xM (in microllters) o~ 10 m~lml RNase (haat treated) was : then added and allowed to digest tor 30 minutes at room t~mperatur6 or overnight at 4C. 0.615xM ~in microliters) ol a solution of Proteinase K
(20mg/ml) was then added and incubated at 55C tor three hours. DNA
was extract~d with 1/2 volume ot neutralized phenol and 1/2 volume ot 2 S chlorotorm and precipitated with two volumes ot ethanol.
DNA ~ resuspended in 0.415xM (in milliliters) ot water, and 0.05xM millilitors ot 5 M NaCI and 0.155xM milliliters ot 50% (w/v) polyothyleneglycol (PEG) (molecular weight 6û00-8000) were added, incubat~d on ice water tor one hour and precipitated by centritugation tor 3 0 15 minutos at 7,000 x 9. The DNA was r~suspended in 0.04xM milliliters ot watsr and 1/10 volume ol 3M sodium acetate and extracted with 1/2 volum~ ot n~utralized phenol and 1/2 volume ot chlorotorm and prccipitated with two volumes ol ~thanol~ The purified DNA was rssuspended in 0.0476xM milliliters ot deionized H20. The DNA
3 5 concentration was determined by tluorometry.

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rCJ/US~)3/1)1?1X
WO g3~17128 ~, 210 l~80 Finally, the purified DNA was disrupted into small tragmen1s o~
approximately 300 base pairs by sonication using a i3ranson Sonitier 450 (Danbury, Connecticut). This siz~ ot fragm~nts has been empirically d~termin~d to be the optimum tor DNA prcbes used for in situ S hybridization. Four milligrams ot th~ purified plasmid DNA pr0pared above was resuspended in 2 mls ot water and imm~rssd in a dry ica/ethanel 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 bonom of the tube. Sonication was carried out at an output I n power of 25-30 watts, discontinuously, with an 80% duty cyle (on 80% ot time, off 20% ot time), tor a period ot 5 minutes. Following sonication, the DNA was precipitated by the addition ot 0.2 ml ot 3 M sodium acetate (pH
5.5) and 4 ml of ethanol. The precipitate w~s recovered by centrifugation tor 5 minutes at 8,000 x 9 and vacuum dried.
; 15 Ex~m~e 2. ~isulfit~ Cata!yzed Transarnin~iQn Q~ Di~
~' DNA obtained by the m~thod o~ Example 1 was transaminated by the addition of ethylenedlamine to the C4 carbon atom o1 tha base cytosins. This rsaction is cata~yzed by sodium bisu~fite. To prepare the 2 0 bisulfit~ bufter, 1.7 ml ol ~uming HCI was slowly added to 1 ml doionized H20 on ice. 1 ml tresh elhylenediamine (Si~ma cat. #E-4379) was then slowly added on ice. A~ter dissolution o~ the ethylenediamine, the solution was warmed ~o room ~amperature and 0.475 9 sodium metabisul~ite (Aldrich Ca~. ~25,555-6) was added. Fuming HCI was then 2 S slowly added to the bisulfite mixture until the pH reached 7Ø Deionized wa~er was addsd lo a final volume o~ 5.0 ml. To transaminate DNA, 1 milli~ram ol sonicated DNA was rssuspended in 0.3 ml H20. The DNA
was dena~ured by boiliny at 100C lor S minutes Ihen quickly chilled in an ice watsr bath. The transamination roaction was initiated by the 3 0 addition ot 0.3 ml of this DNA solution to 2.7 ml ot bisulfits buHer, and ~he reaction was incubated a~ 37C ~or 2 days. The DNA solution was desalted by routine dialysis against 5-1û millimolar sodium borat~ (pH
8.0). Att~r dialysis, 0.3 ml ot 3 M sodium acetate (pH 5.5) was added to th~ dialysate. The aminatod DNA was procipitated with 2.5 volumes ot 3 5 etllanol and recovered atter centrifugation at 8,000 x 9 tor 10 minutes.

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WO 93/17128 P~/US93/1)1718 7 ~
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The pellets were vacuum dried and r~hydrat~d at a conc~ntration of 3 mglml DNP~. This solulion was stored at -~0C until usa, :~;
.1 Exanl~ 3; PreearaIiQ~Iitro~h~t A solution ot 20 mM -amino-n-caproic acid was prepared by - ~ adding 2 62 9 of this compound to 20 ml water containing 40 mmol ; sodium bicarbonat~. This solution was mixed with 20 ml of a 20 mM
- solu~ion ot Sange~s reagent (2,4 dinitro-tluorobenzene) and allowed to 10 s~and at room temperature for 1 hour. The mixture was then gently .~ ~ h~ated, which caused the solution to turn yellow and a small amount ot th~ dissolved sodium bicarbonate to precipitate. This precipitate was re-. dissolved by the addition o~ a sufficient quantity ot concentrated HCI and than lett at 4C to induce cryslallization. The c~stals were collected in 15 vacvo and washed with watsr to yisld 4.2 9 of yellow crystalline E-dinitrophenylamino ncaproic acid (DNP-NGA).
DNP NCA was activat~d by esteritication to 3-sulfo-N-hydroxysuccinimid~ as follows. 0.594 ~ of DNP-NCA, 0.468 9 dicyclohexylcarbodiimlde and 0.434 ~ ot 3-sulto-N-hydroxysuccinimide 2 0 were vigorously stirrsd In 7 ml dimethylformamids at room temperature overnight. This reaction was dstsrmined to have gone > 90% to completion by thin layer chromatography. The mixture was cooled to 0C
and slirred for an additlonal hour. The mixturs was then filtcred and the y011Ow solution evaporated lo a IhiCk yellow oil which did not crystallize.
2 5 This oil was slirred with 50 ml elhano~ to yield 0.996 9 ol a tine yellow powder which was collected by filtration and washed with ethanol. This compound is 6~N-(2,4 1lnitroph~nylamino)caproic acid O-(N-hydroxysuccinimidc)-3 sulto"ale ~sodium salt) and will bs reterred to tor ths purposes ot thls invention as S-NHS DNP.
blins~ nY~atiyQd E arniD~
Chromosome-specitic DNA o~ average length of about 300 bp propar~d by tho method ot Example 1 was derivatized by bisulfite 3 5 catalyzod transamination with ethylensdiamin6 as described in Example 2. A solution ot aminated DNA (100 1l9 total DNA) in a plastic 1.5 ml centrifuge tube was evaporaled under reduced pressure. 0.5 ml ot 0.2 M
3-lN-morpholino] propane sultonic acid (MOPS) buffer and then 100 '!

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' ' WO 93/17128 PCr/US93/Olr 8 - 21078~0 microliters ot S-NHS-DNP (30mg/ml N,N-dimethy~ormamide) was added to the residue and the mixlure was incubated overnight at 25C, DNP-Labe16d DNA was precipitated by the addition ot 60 ~11 ot 3 M sodium a~tata (pH 5.5) followed by 1.5 ml of ic~ cold etha~ol and tha mixture 5 was incubated for at least 2 hours at -20C. The solution was subjected to centrifugation tor 10 minutes at 10,000 x 9. The DNA pellet was washed twice with 0.6 ml ot ice cold ethanol and then dissolved in 100 ,ul ot sterile water. Two Sephadex G-50 Select D chromatography columns (5 Prime -> ~ Prime, Inc.) with a bed volume ot 0.8 ml were prepared. 50 10 ,ul ot the dissolved pellet was applied ~o each column and centrifuged tor 4 minutes at 10,000 x 9. 10 1ll ot the purifieci DNA was diluted with 490 1ll of 20mM NaOH and the optical density was determined at 260 nm to assess DNA concentration. The purified DNA was dilut~d with water to provide a working concentration ot DNA ot 100 micrograms per ml.

,Exzm~_~repa~ of theo~hviline-~-N-~5 hyg~QxvDen~-lamino~-o-succ;~oyl-O~-tN~ -s~ sI~
A solution ot 5.16 grams ot 8-bromotheophylline and 5.15 grams ol 5-amino-1 pentanol in 18 ml ot ~xylene was refluxed for 18 hours.
20 The reaction solu~ion was allowed to cool and upon cooling to room temperature lormed a solid~quid mixture. The xylene was decanted and the solid was washed with pentane (3x40 milliliter). Tha resulting solid was stirred in 20 milliliter ot water tor 30 mlnutes and tiltered to collect thesolid material. The colleaed solid matarial was washed with water (3x20 2 5 milliliter), and dried to provids 3.73 grams ot 8-(5-hydroxypentylamino)-theophylline. To a solution ot 1.12 ~rams ot 8-(5-hydroxypentylamino)-theophylline in 1S milliliter ot anhydrous N,N-dimethyltormamide (DMF) was addad 0,516 gram ot succinic anhydride and 0.2 gram ot 4-dimathylamino pyridino (DMAP). Ths mixture was stirred with a magnetic 3 0 stirr~r ovornight al room temperaturs under anhydrous conditions. The colorless solid that precipitated was collected by vacuum tiltration, washod with dichloromothane and air dried to provide 1.20 gram ot theophylline-8-N-(5-hydroxypentylamino)-O-succinic acid ester. This solid was recrystallized trom a boiling 1:1.propanoUwater mixturs and 3 5 dried over calcium sultate to provida a c~lorless solid. To a solution ot 0.725 gram ot theophylline-8-N-(5-hydroxypentylamino)-O-succinic acid ester in 15 ml ot anhydrous dimethyltormamide was added 0.414 gram ot ,~

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I'CI / IJS93/01718 21~ 8 0 3-su~fo-N-hydroxysuccinimlde (Sullo-NHS). A solution o~ 0.48B gram o?
dicyclohexyl-carbodiimid~ (DCC) in 2 milliliter ot anhydrous DMF was added to the abov~ solution. The resulting mixture was stirred with a - ma~netic stirrer al room t~mp~ratur~ tor 16 hours. The r~action mixtur~
5 was cool~d in an ice bath for 1 hour, th~n filtered under vacuum to r~move dicyclohexylurea. The filtrate was evaporated jn vacuo (2 torr, 30C) to provide a viscous colorless oil. This oil was troat~d with 40 milliliter ot anhydrous ethanol and a solid material precipitated. Th~
precipitated material was collected by fi~tration and dried over anhydrous 10 calcium sulfate to provide 0.450 gram of theophylline-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sultosuccinimidyl)) est0r, (NHS-theophylline).
EX-~m~ 6 DNA l,a~inQ - Ib~b~i~
Chromosome-specific DNA probes to human chromosome 4 of 15 average length of about 30û bp obtained by the procedure of Example 1 were derivatized with the bisuliit~ catalyzed transamination with ethylenediamine as dsscnb~d in Example 2. Approximately 5% of the basas were aminat~d. A solution oi aminated DNA (100 micrograms total DNA) in a plastic 1.5 millililer cenlntuge tube was evaporat~d under 20 reduced pressure. 0.5 millililer ol 0.2 molar 3-[N-morpholino3 propane sulfonic acid (MOPS) butter and then 100 microliters of NHS-theophylline (26 milligramlmillili~er N,N-dimethyltormamide) was added to the residue and the mi~nure was incubated overnight at 25C. 50 microlitqrs ot 3 M sodium acetate (pH 5.5) were added followed by 1.5 2 5 milliliter of Ice cold ethanol and the mixture was incubated for at least 2 hours at 20C. The solution was subjected to centritugation tor 10 minutss at 10,000 x 9. The pellet was washed twice with 0,6 milliliter of ic0 cold ethanol and then dissolvad in 100 microliters of sterile water.
Two Ssphadex G 50 Select D chromatography columns (5 Prime ~ 3 30 Prims, Inc.) with a bed volume ol 0.8 milliliter were prepared. 50 microlitors ot the dissolved pellet was applied to each column and csntrituged tor 4 minutes at 10,000 x 9. 10 microliters ot the puritied DNA
was diluted with 490 microliters ot 20 millimolar NaOH and the optical dsnsity was determined at 260 nm to assess DNA concentration. Th6 3 5 purified DNA was diluted with water to provide a working concentration ot DNA ot 100 micrograms per milliliter.

~: 23 .;

07~8~ ~
, ~xamDI~ 7 n~ ; s-(and-~) Gar~oxvt~am~
su~;inirnidvl ~st~r ~
Chromosome-specitic DNA probes to human chromosomes 1 and 4 ot averaga length ot about 300 bp obtain0d by ~he procedure ot Example 1 were derivatized by the bisultite catalyzed transamination with ethylenediamine as described in Example 2. Approximately 5% ot th~
bases were aminated. A solution ot aminat0d DNA (50 micrograms total DNA) in a plastic 1.5 milliliter centrifuge tube was evaporated under reducsd pressure. To this solution was added 377 microliter of 0.2 Molar 3-lN-morpholinol propane sulfonic acid (MOPS) pH 7.4 buffer. Twenty-two and eight tenths microliters of CTMR (5-(and-6)-carboxytetramethylrhodamine, succinimidyl estsr, 50 millimolar in N,N-dimethylformamide) was added to ths transaminated DNA and the mixture was stirred overnight at -25C (approximately 18 hours). The excess tluorophore was separated trom th0 labeled DNA by ethanol precipitation. The preupltated DNA peliet was dissolved in sterile water, then passed over a Sepnadex G-25 column that was 28 centimeters high with an internal diameter of 1 centlmeler. The desired fraction (!he column void volume) was elutsd ~th water and dried to reduce the total volume. A second elnanol preapltallon o~ the DNA completed the purification.

Example1 Q~a~ o~ T!~mlnaled Chromosor~ ~2_cifi~ pN~, Probe~ with th~ Fluoroonore5-!and 61 car~oxv~1uor~uccinimidyl 2 5 esî~r (CFI~

Transaminated DNA probes ob~ained by ~he method ot Example 2 were conjugated with 5-(and-6)-carboxytluorescein, succinimidyl ester (CFI). Fitty micrograms ot transamlnated DNA were dried and then 3 0 rosusp6nded in 37-? microliîers ol 200 mM MOPS, pH 7.4. Twenty-two and ~ight t~nths microliters ot 50 mM solution ot 5-(and-6)-carboxytluorescein, succinimidyl ester, (CFI) in N,N-dimethyltormamide (a 150-told molar excess) was added to the transaminated [)NA. This reaction proceeded with stirring in darkness at room tomperature 3 5 overnight (approximately 18 hours). The excess tluorophore was separated trom the labeled DNA tirst by an ethanol precipitation. The precipitaled material was resuspended in water and passed over a Sephadex G-25 column that was 2B cm high with an internal diameter ot .

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- - ~Vo 93/17128 I~CI /US93/01718 2~7~
1 cm. The d~sirad fraction (th~ column void volum~) was elut~d in water .- and dried to reduc0 the total volume. A sacond ~thanol pr~cipitation of th~ label~d DNA completed the purification. An absorbanoe spectrum showed that 1.6% of the bases wer~ labeled.

Exaq~RlQ9 PreDaration ot Meta~ e SDreads SupB13 cells (CML line with multiple translocations case #1053~) w~ra made available by Michelle LeBeau, University of Chicago. Cells were reseeded into 25 cubic-centimeter tlasks containing 80% RPMI
I 01640 media (Gib~o catalog #320-1875), 20% ~etal calf senJm, 100 units Penicillin/Strsptomycin, and 10 millimolar HEPES buffsr. Cell growth was monitored by counting and cells were refed every 3 or 4 days. When cells had achieved oplimal conc~ntration, 0.2 ml 10 microgramslmilliliter Colicimed (Gibco catalog#890-1 145-l) was added to each flask, and then 15 held for 1 hour at 37C in a 5% C02 incubator. Cells were harvestad by centrifugation and resuspended in 5 milliliters 75 millirnolar KCI, then held tor 10 minutes at 37C. Cells were again harvested by centritugation, and all but 0.2 millili~er of the KCL supernatant was removed. The cells were dlspersed, Ihen fixed slowly in 10 milliliters 3t1 20 methanoUacetic acid,.and held on ice lor 15 minutes. Cells were har'vesled by centriluga~ion and placed in 5 milliliters Iresh m~thanollac~tic acid, and held another 15 minutes on ice. Cells were harvested, resuspended in methanol acetic acid, and placed dropwise onto precleaned glass slides and dried.
2 5~amRIQ~ Situ Hyhndi7atio~ ProtOCQl The coupled assay was lestsd by in si~u 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 Iocate areas containing nuclei and metaphase spreads. Ou~lines ot ~he 3 0hybridization target area, and identi~ication rnarks were made on the r~vsrse side o~ the slide with a diamond scribe. Belore hybridization, the target DNA on the slide was denatured by immersion tor 2-10 minutes in a solution o~ 70% tormamide/o~3 M NaCU30 millimolar sodium citrate, pH
7.6~7.B at 70 C. Following denaturation, the target DNA was placed in a ~' 3 570/0 ethanoUwater bath and agitated to remove the ~ormamide solution.
The wash step was repeated by passing the slide through 70%, 85% and .~
~' Wo 93/17128 2 1 0 7 8 8 0 PC~I/US93/O,~lX

100% ethanol (ambient temperature, 2 minutes each). The hybridizalion mixture consisted ot 50% tormamide/0.3 molar NaCI/30 millimolar sodium cilrata, pH 7Ø Human placental DNA (2.25 micrograml10 microlit~r) was used as Ih0 blocking DNA. The th~ophylline lab~ d WCP l and the DNP labelled WCP 4 were each added to a concentration ot 10 nanogramlmicroliter. The total hybridization volume was 10 microlit~r. The hybridization mixture was denatured tor 5 minutes at 70 C, ~hen addGd to the slids over the area which contained the target DNA. A coverslip was placed on the hybridization mixture, and the l O 0dges were sealed with nJbber c0ment. 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 ~0% formamide/0.3 M NaCI/3 millimolar sodium citrate. pH 7.0 at 45 C. The slide was ~hen washed 5 I S minutes in 0.3 M NaCI/3 millimolar sodium citrate and 5 minutes in 0.1 M
sodium phosphate/0.1% NP40 (PN Buf1er) each at 45 C. The slide was washed twice in PN bufter al room temperature, 2 ~ninutes each-wash.
The slide was incubated tor 20 minutes in anti-dinitropher~ol (rabbit) diluted 1:250 in PNM buHer (PN buffer with 5% nonfat dry milk). The slide was washed 3 limes in PN bufler al ambient temperature for 2 minutes aach time. The slide was ~hen incubated for 20 minutes in glucose oxidase-anti rabbit conjugata, and again wash~d 3 times in PN
buffer. The slide was tinally incubatsd lor 20 minut~s in horseradish p0roxidase-anti theophylline conjugate and washed a final 3 times in PN
2 5 butler, with trash buf1er used tor each washing step.
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~m~Q l l . Color D~vel~Dme~lt and Resll~
A modltication of the commercially available Vector TMB
'! (tslramethylbcnzidine) peroxidase kit was used in lhe color development Specitically, hydrogen peroxide was omitted trom the substrate - 30 preparation and 100 microlitsrs o~ l ~iolar glucose was substituted ther~in. Ths slids bearing the prapared, hybridized m~taphasr~ spraad was incubat~d l hour with the chromog~nic reagent, rinsed briefly under ;~ a g~ntb stream ot distilled water, then air dried.
. . , Thc TM~ treated metaphase spread was counterstained for 5 3 5 minutes in treshly prepared, nltered Giemsa. The slide was rinsed under a gentle stream ot distilled water. The slide was then dned with an air or `~ 2 6 .

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nitrogen jet. The stainea metapnase spread was then examined in a microscope .

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

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Claims (10)

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 anattached first label, and comprises high and moderate complexity DNA
sequences which are complementary lo 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 byimmunological 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 immunologically attaching to first labels, and second antibodies which are conjugated to second ISPM's, and capable of immunologically attaching to second labels, or (2) first antibodies which are conjugated to first ISPMs, and capable of immunologically attaching to first labels, second antibodies capable of immunologically attaching to second label, and third antibodies which are conjugated to second ISPMs, and capable of immunologically attaching to said second antibodies, or (3) first antibodies capable of immunologically attaching to first label, second antibodies which are conjugated to second ISPMs, and capable of immunologically 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 fadedor otherwise non-detectable, comprising the steps of;
(a) contacting fluorescent label portion of fluorescence labelled hybridized chromosomes wish a signal producing moiety, wherein said moiety is directed to florescence labelled chromosomes by immunological means comprising antigen/antibody or antibody/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 14, 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 firstprobe 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.