CA1301605C - Methods and compositions for chromosome specific staining - Google Patents

Abstract

METHODS AND COMPOSITIONS FOR
CHROMOSOME SPECIFIC STAINING

ABSTRACT OF THE DISCLOSURE

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

Description

METHODS AND COMPOSITlONS FOR
i CHROMOSOME-SPECIFIC STAlNlNG

BACKGF;OUND OF THE INVENTION
S The United States Government has rignts in tnis invention pursuant to Contract No. W-7405-ENG-48 between tne U.S. Department of Energy and the University of California, for the operation of Lawrence Livermore National La~oratory.
The invention relates generally to the field of cytogenetics, and more particularly, to methods for identifying and classifying chromosomes.

?3~)S

Chromosome abnormalities are associated With genetic disor~ers, degenerative diseases, and exposure to agents known to cause degenerative diseases, particularly cancer, German, "Studying ~luman Chromosomes Today," Ameri-can Scientist, Vol. 58, pgs. 182-201 (1970); Yunis, "The Chromosomal Basis of Human Neoplasia," Science, Vol. 221, p95. 227-236 (1983); and German, "Clinical Implication of Chromosome Breakage," in Genetic Damage in Man Caused by Environmental Ayents, Berg, Ed., pgs. 65-86 (Academic Press, New York, 1979). Chromosomal abnormalities can ~e of three general types: extra or missing individual chro-mosomes, extra or missing portions of a chromosome, or chromosomal rearrangements. The third category includes translocations (transfer of a piece from one chromosome onto another cnromosome), and inversions (reversal in polarity of a chromosomal segment).
Detectable cnromosomal abnormalities occur with a fre~uency of one in every 250 human births. Abnormalities that involve deletions or additions of chromosomal material alter the gene balance of an organism an~ generally lead to fetal deatn or to serious mental physical defects.
Down's syndrome is caused by having tnree copies of chro-mosome 21 instead of tne normal 2. This syndrome is an example of a condition caused by abnormal chromosome 25 number, or aneuploidy. Cnronic myelogeneous leukemia is OS

associated with the exchange of chromosomal material between chromosome 9 and chromosome 22. The transfer of chromosomal material in this leukemia is an example of a translocation. Clearly, a sensitive method for detecting chromosomal abnormalities would be a highly useful tool for genetic screening.
Measures of cnromosome breakage and other aberra-tions caused by ionizing radiation or chemical mutagens are widely used as ~uantitative indicators of genetic damage caused by such agents, Biochemical Indicators of Radiation In3ury in Man (International Atomic Energy Agency, Vienna, 1971); and Berg, Ed. Genetic Damage jn Man Caused by Envi-ronmental Agents (Academic Press, New York, 1979). A host of potentially carcinogenic and teratogenic chemicals are widely distributed in the environment because of industrial and agricultural activity. These chemicals include pesti-cides, and a range of industrial wastes and by-products, such as halogenated nydrocarbons, vinyl chloride~ benzene, arsenic, and tne like, Kraybill et al., Eds., Environmental Cancer (Hermisphere Publishing Corporation, New York, 1977~. Sensitive measures of chromosomal breaks and other abnormalities could form the basis of improved dosimetric and risk assessment methodologies for evaluating the con-sequences of exposure to such occupational and environ-mental agents.

Current procedures for genetic screening and bio-logical dosimetry involve the analysis of karyotypes. A
karyotype is a collection of indices whicn characterize the state of an organism's chromosomal complement. It includes such things as total chromosome number, copy number of individual chromosome types (e.g., the number of copies of cnromosome X), and chromosomal morpnology, e.g., as measured by length, centromeric index, connectedness, or the like. Chromosomal abnormalities can be detected by examination of karyotypes. Karyotypes are determined by staining an organism's metaphase, or condensed, chromo-somes. Metaphase chromosomes are used because, until recently, it has not been possible to visualize nonmeta-phase, or interphase chromosomes due to their dispersed condition in the cell nucleus.
The metaphase chromosomes can be stained by a number of cytological techniques to reveal a longitu-dinal segmentation into entities generally referred to as bands. The banding pattern of each chromosome within an organism is unique, permitting unambiguous chromosome identification regardless of morpnological similarity, Latt, "Optical Studies of Metaphase Chromosome Organi~a-tion," Annual Review of Biophysics and Bioengineering, Vol. 5, pgs. 1-37 (1976). Adequate karyotyping for detecting some important chromosomal abnormalities~ sucn 1 3~ 0S

as translocations and inversions requires banding analy-sis. Unfortunately, such analysis requires cell cultur-ing and preparation of high quality metaphase spreads, w~ic~ is extremely difficult and time consuming, and almost impossible for tumor cells.
~he sensitivity and resolving power of current methods of karyotyping, are limited by the lack of stains that can readily distinguish different chromosomes having hignly similar staining characteristics because of simi-larities in such gross features as size, morphology, and/or DNA base composition.
In recent years rapid advances have taken place in the study of chromosome structure and its relation to genetic content and DNA composition. In part, the prog-ress has come in the form of improved methods of gene mapping based on the availability of large quantities of pure DNA and RNA fragments for probes produced by genetic engineering techniques, e.g., Kao, "Somatic Cell Genetics and Gene Mappings," International Review of Cytology, Vol.
85, pgs. 109-146 (1983), and D'Eustacnio et al., "Somatic Cell Genetics in Gene Families," Science, Vol. 220, pgs. 9, 19-924 (1983). The probes for gene mapping comprise labeled fragments of single stranded or double stranded DNA or RNA which are hybridized to complementary sites on chromosomal DNA. The following references are representa-OS

tive of studies utilizing gene probes for mapping: Harper et al. "Localization of the Human Insulin Gene to the Distal End of the Short Arm of Chromosome 11," Proc. Natl.
Acad. Sci., Vol. 78, pgs. 4458-4460; Kao et al., "Assign-S ment of the Structural Gene Coding for Albumin to Cnromo-some 4," Human Genetics, Vol. 62, pgs. 337-341 (1982);
Willard et al., "Isolation and Characterization of a Major Tandem Repeat Family from tne Human X Chromosome,'` hucleic Acids Research, ~ol. 11, pgs. 2077-2033 (1983); and Falkow et al., U. S. Patent 4,358,535, issued 9 November 19~2, entitled "Specific DNA Probes in Diagnostic Microbio'ogy."
The hybridization process involves unravelling, or melting, the double stranded nucleis acids by heating, or other means. This step in the hybridization process is sometimes lS referred to as denaturing tne nucleic acid. When tne mix-ture of probe and target nucleic acids cool, strands naving complementary bases recombine, or anneal. When a probe anneals with a target nucleic acid, the probe's location on tne target can be detected by a label carried by the probe. When the target nucleic acid remains in its natural biological setting, e.g., DNA in cnromosomes or cell nuclei (albeit fixed or altered by preparative techniques) tne hybridization process is referred as in situ hybridization.

Use of hybridi2ation probes has been limited to identifying tne location of genes or known DNA sequences on cnromosomes. To this end it has been crucially impor-tant to produce pure, or homogeneous, probes to minimize hybridizations at locations other tnan at tne site of interest, Henderson, "Cytological Hybridization to Mammalian Chromosomes," International Review of Cytology.
Vol. 76, pgs. ~-46 (1982).
Manuelidis et al., in 'ICnromosomal and Nuclear Distribution of tne Hind III l.9-KB Human DNA Repeat Seg-ment," Chromosoma, Vol. 91, pgs. 28-38 (1984), disclose the construction of a single kind of DNA probe for detecting multiple loci on chromosomes corresponding to members of a family of repeated DNA sequences.
Wallace et al., in "The Use of Synthetic Oligo-nucleotides as Hybridization Probes. lI. Hybridization of Oligonucleotides of Mixed Sequence to Rabbit Beta-Globin DNA, "Nucleic Acids Research, Vol. 9, pgs. 879-894 (1981), disclose tne construction of synthetic oligonucleotide probes having mixed base sequences for detecting a single locus corresponding to a structural gene. The mixture of base sequences was determined by considering all possible nucleotide sequences whiCh could code for a selected sequence of amino acids in the protein to wnich the struc-tural gene corresponded.

1 ~3~?1~C?5 Olsen et al., in "Isolation of Uni~ue Se~uenceHuman X Chromosomal Deoxyri~onucleic Acid," Biochemistry, Vol. 19, pgs. 2419-2428 (1980), disclose a method for isolating labeled unique se~uence human X chromosomal DNA
by successive hyDridizations: first, total genomic human DNA against itself so that a uni~ue se~uence DNA fraction can ~e isolated; second, tne isolated uni~ue sequence human DNA fraction against mouse DNA so tnat nomologous mousethuman sequences are removed; and finally, the unique se~uence human DNA not homologous to mouse against tne total genomic DNA of a human/mouse hybrid wnose only human chromosome is chromosome X, so that a fraction of unique sequence X cnromosomal DNA is isolated.

SUMMARY OF THE INVENTION
The invention includes methods and compositions for staining chromosomes. ln particular, chromosome specific staining reagents are provided which comprise heterogeneous mixtures of labeled nucleic acid fragments having substantial portions of substantially complementary base se~uences to the chromosomal DNA for which specific staining is desired. The nucleic acid fragments of the heterogenous mixtures include double stranded or single stranded RNA or DNA. Heterogeneous in reference to tne mixture of la~eled nucleic acid fragments means that the staining reagents comprise many copies each of fragments having different base compositions and/or sizes, such that application of tne staining reagent to a cnromosome results in a substantially uniform distribution of fragments hy~ridized to the cnromosoma) DNA.
'~substantial proportions" in reference to tne basic sequences of nucleic acid fragments that are comple-mentary to chromosomal DNA means tnat the complementarity is extensive enougn so tnat tne fragments form stable hybrids with the chromosomal DNA under standard hybridiza-tion conditions for tne size and complexity of the frag-ment. In particular, the term comprehends the situation where the nucleic acid fragments of tne heterogeneous mixture possess regions having non-complementary base sequences.
As discussed more fully below, preferably tne heterogeneous mixtures are substantially free from so-called repetitive sequences, botn tne tandem variety and the interspersed variety (see Hood et al., Molecular Biology of Eucaryotic Cells (Benjamin/Cummings Publishing Company, Menlo Park, California, 1975) for an explanation of repetitive sequences). Tandem repeats are so named because they are clustered or contiguous on the DNA mole-cule wnicn forms tne backbone of a chromosome. Mem~ers of this class of repeats are also associated with well-defined s regionS of tne cnromosome, e.g., the centromeric region.
Thus, if these repeats form a si2able fraction of a chromo-sorne, and are removed from tne neterogeneous mixture of fragments employed in the invention, perfect uniformity of staining may not be possible. This situation is compre-hended by the use of the term "su~stantially uniform" in reference to tne heterogeneous mixture of labeled nucleic acid fragments of the invention.
lt is desirable to disa~le the nybridization capacity of repetitive sequences because copies occur on all the cnromosomes of a particular organism; thus, their presence reduces the chromosome specificity of tne staining reagents of tne invention. As discussed more fully below, hybridization capacity can be disabled in several ways, e.g., selective removal or screening of repetitive sequences from chromosome specific DNA, selective blocking of repetitive se~uences by pre-reassociation witn comple-mentary fragments, or the like.
Preferably, the staining reagents of tne invention are applied to interpnase or metapnase chromosomal DNA by in situ hy~ridization, and tne cnromosomes are identified or classified, i.e., karyotyped, by detecting the presence of tne label on the nucleic acid fragments comprising the staining reagent.

The invention includes chromosome staining reagents for tne total genomic complement of cnromosomes, staining reagents specific to single chromosomes, staining reagents specific to subsets of cnromosomes, and staining reagents specific to subregions within a single chromosome.
The term "cnromosome-specific," is understood to encompass all of these embodiments of tne staining reagents of the invention. Tne term is also understood to encompass stain-ing reagents made from both normal and abnormal chromosome types.
A preferred metnod of making tne staining reagents of the invention includes isolating chromosome-specific DNA, cloning fragments of tne isolated cnromosome-specific DNA to form a heterogeneous mixture of nucleic acid frag-ments, disabling the hybridization capacity of repeatedsequences in the nucleic acid fragments, and labeling the nucleic acid fragments to form a heterogeneous mixture of labeled nucleic acid fragments. As described more fully ~elow, the ordering of the steps for particular embodiments varies according to the particular means adopted for carry-ing out the steps.
The preferred metnod of isolating chromosome-specific DNA for cloning includes isolating specific chro-mosome types by fluorescence-activated sorting.

~ 3~16~)5 - The present invention addresses problem5 asso-ciated with karyotyping chromosomes, especially for diag-nostic and dosimetric applications. In particular, tne invention overcomes problems which arise because of the lack of stains tnat are sufficiently chromosome-specific by providing reagents comprising heterogeneous mixtures of la~eled nucleic acid fragments that can ~e hy~ridized to the DNA of specific chromosomes, specific subsets of chro-mosomes, or specific subregions of specific chromosomes.
The staining techni~ue of the invention opens up the pos-sibility of rapid and highly sensitive detection of chro-mosomal abnormalities in botn metaphase and interphase cells using standara clinical and laboratory equipment.
It has direct application in genetic screening, cancer diagnosist and biological dosimetry.

OS

DETAlLED DESCRIPTlON OF THE lN~E~TI~N
The present invention includes compositions for staining individual chromosome types and methods for making and using the compositions. The compositions comprise heterogeneous mixtures of labeled nucleic acid fragments. The individual labeled nucleic acid fragments making up tne heterogeneous mixture are essentially stan-dard hybridization probes. That is, eacn chromosome-specific staining composition of the invention can De viewed as a large collection of hybridization probes to unique sequence regions of a specific chromosome. ln fact, the preferred metnod of making tne compositions of tne in~ention entails generating a heterogeneous mixture on a fragment-by-fragment basis by isolating, cloning cnromosomal DNA, and selecting from the clones hybridi-zation probes to unique sequence regions of a particularchromosome. The precise number of distinct labeled nucleic acid fragments, or probes, comprising a hetero-geneous mixture is not a critical feature of the inven-tion. However, in particular applications, a trade-off may have to be establisned ~etween the number of distinct fragments jn a heterogeneous mixture and the degree of nonspecific DaCkgrOund staining: Where the tendency for nonspecific background staining is hign~ giving rise to low signal to noise ratios, it may be necessary to reduce ?1~()5 tne number of distinct fragments comprising tne netero-geneous mixture. On tne other hand, where nonspecific background staining is low, tne number of distinct frag-ments may be increased. Preferably, tne num~ers of dis-tinct fragments in a neterogeneous mixture is as hig~ aspossible (subject to acceptable signal to noise ratios) so tnat staining appears continuous over the ~ody of tne chromosomes.
Another constraint on the number of distinct fragments in the heterogeneous mixture is solubility.
Upper bounds exist witn respect to tne fragment concentra-tion, i.e., unit length of nucleic acid per unit volume, tnat can ~e maintained in solution. Thus, if fragments of a given average length are used at a given concentration (fragment per volume), tnen the num~er of different such fragments that can comprise the heterogeneous mixture is limited.
In one preferred embodiment where tne heterogen-eous mixture is generated on a fragment-by-fragment ~asis, tne cnromosomal DNA is initially cloned in long se~uences, e.g., 35-45 kilobases in cosmids, or like vector. After amplification the inserts are cut into smaller fragments and ~abeled for formation to a heterogeneous mixture. In tnis embodiment, the chromosomal binding sites of tne fragments are clustered in the chromosomal regions comple-1 ~3~ )5 mentary to the cloned "parent" nucleic acid sequence.
F 1 u o r escent signals from SuCh clusters are more readily detected than signals from an e~uivalent amount of label dispersed over the entire chromosome. In this embodiment, the clusters are substantially uniformly distri~uted over tne cnromosome.
Repetitive sequences, repeated se~uences, and repeats are used interchangeably tnroughout.

I. lsolatiDn of Chromosome-specific DNA and Formation o DNA raqment Librartes.
.
The first step in the preferred method of making the compositions of tne invention is isolating chromosome-specific DNA. This step includes first isolating a suffi-cient quantity of the particular chromosome type to wnich the staining composition is directed, then extracting the DNA from tne isolated chromosomes. Here "sufficient ~uan-tity" means sufficient for carrying out subsequent stepsof tne method. Prefera~ly, the extracted DNA is used to create a chromosome-specific library of DNA inserts wnich can be cloned using standard genetic engineering tech-ni~ues. The cloned inserts are then isolated and treated to disable the hybridization capacity of repeated sequences. In this case, Nsufficient quantity" means ènough for tne particular method used in constructing the DNA insert library.

` 13~ 05 j Several met~ods are available for isolating par-ticular chromosome types. For example, a technique for isolating human cnromosome types involves forming hybrid cell lines from numan cells and rodent cells, parti~ularly mouse or hamster cells. e.g., see Kao, ~Somatic Cell 6enetics and Gene Mapping,~ International Review of Cytology. Vol. 85, pgs. 109-146 ~1983), for a review.
Human c~romosomes are preferentially lost by tne ny~rid cells so tnat nybrid cell lines containing a full comple-ment of rodent chromosomes and a single human cnromosome can be selected and propagated, e.g., Gusella et al., ~lsolation and Localization of DNA Seg~ents from Specific Human Cnromosomes,U Proc. Natl. Acad. Sci., Yol. 77, pgs.
2829-2833 (1980). Chromosome specific DNA can tnen be isolated by tecnni~ues disclosed by Schmeckpeper et al., ~Partial Purification and characterization of DNA from ~uman X C~romosome,~ Proc. Natl. Acad. Sci., Yol. 76, pgs.
6525-6528 (1979); or Olsen et al., ~lsolation of Unique Sequence Human X Cnromosomal Deoxyri~onucleic Acid,"
Biochemistry, Vol. 19, pgs. 2419-2428 (1980). Briefly, sheared total human DNA is hybridiZed against itself on hydroxyapatite under conditions that allow elution of su~-stantially pure unique sequence total human ~NA from tne hydroxyapatite. ~ne uniQue sequence total human DNA is B
. .

- l 7 tnen reassociated and nick translated to add a label tsee Maniatis et al., Molecular Cloning A La~oratory Manua', Cold Spring Harbor La~oratory, 1982 , pgs. 109-112, for a description of the nick translation technique to add radio-active labels; and Brigato et al., "Detection of ViralGenomes in Cultured Cells and Paraffin-Embedded Tissue Sections Using Biotin-Labeled Hy~ridization Probes,"
~irology, Vol. 126, pgs. 32-50 (1982) for a description of tne nick translation technique to add a biotin label).
Poly(dA) tails are then synthesized to the 3' nydroxyl termini of tne unique sequence DNA by incu~ating witn terminal transferase under appropriate conditions. Tne poly(dA) unique human DNA is then hybridized against rodent DNA to remo~e homologous sequences. The nonhomologous poly(dA) unique human DNA is then hybridized against tne total DNA of the human/rodent hybrid on hydroxyapatite;
double stranded DNA is isolated. (Tnis is tne chromosome-specific uni~ue sequence human DNA.) Tne poly(dA) tail is used to separate tne labeled unique sequence DNA from tne unlabeled unique sequences by binding the polu(dA) tails to oligo(dT) cellulose.
The preferred means for isolating particular chromosome types is by direct flow sorting of metaphase cnromosomes witn or without the use of interspecific hybrid cell systems. Direct sorting is preferred because - - 1 3~ ()5 tnere is considera~le DNA se~uence nomology between rodent and human DNA, WhiCh necessitates additional nybridization steps (e.g., see 01sen et al., cited above), and the nybrid cell lines tend to be unsta~le with respect to retention of t~e ~uman chromosomes.
For some species, every cnromosome can be isolated by currently available sorting tecnniques. Most, but not all, numan chromosomes are currently isolatable by flow sorting, Carrano et al., ~Measurement and Purification of Human Chromosomes by Flow Cytometry and Sorting,a Proc.
Natl. Acad. Sci., Yol. 76, pgs. 1382-1384 (1979). ~hus, for isolation of some human chromosomes, use of tne numan/
rodent hybrid cell system may be necessary. Chromosome sorting can be done by commercially available fluorescence-1~ activated sorting macnines, e.g., Becton Dickinson FACS-II~
or like instrument.
DNA is extracted from the isolated chromosomes ~y standard techni~ues, e.g., Marmur, ~A Procedure for the Isolation of Deoxyri~onucleic Acid from Micro-Organisms,"
~. Mol. Biol., Vol. 3, pgs. 208-218 (1961); or Maniatis et al., Molecular Cloning: A La~oratory Manual (Cold Spring Harbor Laboratory, 1982) pgs. 280-281.

~ * ) Trademark ()S

6eneration of insert li~raries from tne isolated chromosome-specific DNA is carried out using standard genetic engineering techni~ues, e.g., Davies et al., ~Cloning of a Representative Genomic Li~rary of t~e Human X Chromosome After Sorting by flow Cytometry,~ Nature, Yol. 293, p95. 374-376 (1981); Krumlauf et al., UConstruc-tion and Characteri~ation of Genomic LiDraries from Specific Human Chromosomes~ Proc. Natl. Acad. Sci., Yol. 79, pgs. 2971-2975 (1982); Lawn et al., UThe Isolation and Characterization of Linked Delta-and-Beta-Globin Genes from a Cloned Library of Human DNA." Cell, Vol. 15, pgs.
1157-1174 (1978); and Maniatis et al., ~~.olecular Cloning:
A Laboratory Manual,U (Cold Springs Harbor La~oratory, 1982), pgs. 256-308.
In some cases, it is preferable tnat tne nucleic acid fragments of tne heterogeneous mixture consist of single-stranded RNA or DNA. The binding efficiency of single stranded nucleic acid pro~es nas been found to be nigher during in situ nybridi2ation, e.g., Cox et al., ~Detection of mRNAs in Sea Urcnin Embryos by In Situ Hybridization Using Asymmetric RNA ProDes,~ Developmental Biology, Vol. 101, pgs. 485-502 (1984). Standard metnods are usea to generate RNA fragments from isolated DNA frag-2~ ments. For example, a method developed ~y 6reen et al., .

13(~1~0S

descri~e~ in Cell, Yol. 32, pgs. 681-694 (1983), is com-mercialy available from Promega Biotec (Madison, Wl) under B the tradename ~Riooprooe.'' Otner transcription kits suit-able for use with the present invention are available from United States Biochemical Corporation (Cleveland, OH) under the tradename "Genescribe." Single stranded DNA probes can be produced witn tne single stranded ~acteriopnage M13, also available in kit form, e.g. Bethesda Research Labora-tories (Gaithersburg, MD).

Il. Disabling the Hybridization Capacity of ~ 7~ve Sequences.
r As mentioned above, it is desirable to disable the hybridization capacity of repetitive sequences by removal, block, or like means. Repetitive sequences are distributed throughout the genome; most are not chromosome-specific.
Consequently, in spite of the fact that the nucleic acid fragments of the invention are derived from isolated chro-mosomes, tne presence of repeats greatly reduces the degree of Ch romosome-specificity of tne staining reagents of tne invention, particularly in genomes containing a significant fraction of repetitive sequences, such as tne human genome.
Several tecnni~ues are available for disabling the hybridization capacity of repetitive sequences. Highly repetitive DNA sequences can ~e removed from tne extracted chromosome-specific DNA by denaturing and incu~ating the ~e~k :~3~ ()S
-- 2~

extracted DNA against itself or against repetitive sequence-enriched total genomic DNA on hydroxyapatite, or like absorbent, at low Cot values, followed by fraction-ation of dou~le stranded DNA from single stranded DNA.
Hydroxyapatite chromotograpny is a standard tech-nique for fractionating DNA on tne basis of reassociation conditions such as temperature, salt concentration, or tne like. It is also useful for fractionating DNA on the basis of reassociation rate at fixed reassociative canditions, or stringencies. Materials for hydroxyapatite chromotog-raphy are available commercially, e.g., Bio-Rad Labora-tories (Ricn~ond, CA).
Fractionation Dased on resistance to Sl nuclease can also be used to separate single stranded from double stranded DNA after incuDation to particular Cot values.
See Britten et al. "Analysis of Repeating DNA Sequences ~y Reassociation," in Metnods in Enzymology, Yol. 29, pgs.
363-418 (1974), for an explanation of Cot values. Pref-erably, this initial reassociation step is carried out after the extracted cnromosome-specific DNA nas been Droken into pieces and amplified by cloning.
One embodiment of the invention is o~tained by labeling the fragments of the single stranded fraction eluted from tne hydroxyapatite in the initial reassociatjon step. The particular Cot values required to separate i6 miacle repetitive ~nd nignly repetitive se~uenc-es from the chromosome-specific DNA may vary from species to speties because of inter-specific dif~erences ifi tne fraction of the genomic DNA comprising middle ana hi9nly repetitive se~uences. Most preferably in tnis em~odiment, the initial reassociation step is to a Cot value in the range of about a few hundred to a few tnousand.
In addition to self nybridization or hybridjzatjon against repetitive-sequence-enricned total genomic DNA, removal of repeats from fragment mixtures can also be accomplished by hybridiZatiOn against immo~ilized high molecular weignt total genomic DNA, following a procedure described by Brison et al., U6eneral Method for Cloning Amplified DNA by Differential Screening with Genomic lS Probes,~ Molecular and Cellular Biology, Vol. 2, pgs.
578-587 (1982). Briefly, the procedure removed repeats from fragment mixtures in the size range of a few tens of bases to a few hundred bases. Minimally sheared total genomic DNA is bound to diazonium cellulose, or like sup-port. The fragment mixture is tnen hyariaized against tneimmobilized DNA to Cot values in tne range of about 1 to 100. The preferred stringency of tne hybridization condi-tions may vary depending on the ~ase composition of the DNA.

lbU~
^ 23 -The preferred means for disa~ling hy~ridization capacity is selecting unique sequence nucleic acid inserts from a chromosome-specific DNA library. For example, following Benton and Davis, "Screening Lambda gt Recombinant Clones by HyDridization to Single Plaques in situ," Science, Vol. 196, pgs. 180-182 (1977), pieces of cnromosome-specific DNA are inserted into lambda gt ~ac-teriophage or like vector. The phages are plated on agar plates containing a suita~le host ~acteria. DNA from tne resulting phage plaques is tnen trans~erred to a nitrocel-lulose filter by contacting the filter to the agar plate.
The filter is then treated with labeled repetitive DNh so tnat phage plaques containing repetitive sequence DNA can be identified. Those plaques that do not correspond to labeled spots on tne nitrocellulose filter comprise clones which may contain unique sequence DNA. Clones from t~ese plaques are selected and amplified, radioactively la~eled, and hybridized to Southern blots of genomic DNA which has been digested with the same enzyme used to generate tne inserted chromosome-specific DNA. Clones carrying uni~ue sequence inserts are recognized as those that produce a sing7e band during Southern analysis.
Anotner method of disabling the nybridization capacity of repetitive DNA sequences within nucleic acid fragments in~olves blocking the repetitive sequences ~y V~

- 24 _ ~re-reassociation of fragments with fragments of repetitive-sequence-enric~ed DNA, ~y pre-reassociation of the target DNA witn fragments of repetitive-sequence-enricned DNA, or pre-reassociation of both the fragments of the heterogeneous mixture and tne target DNA with repetitive-se~uence-enric~ed DNA. ~ne metnod is generally described by Sealy et al., ~Removal of Repeated Sequences from Hybridization Probes,U Nucleic Acid Research, Yol. 13, pgs. 1905-1922 (198~) ~ he term pre-reassociation refers to a hybridiza-tion step involving the reassociation of unlabeled, repet-itive DNA or RNA with tne nucleic acid fragments of tne heterogeneous mixture just prior to the in Situ hy~ridiza-tion step, or with the target DNA either just prior to orduring tne in situ hy~ridization step. ~his treatment re-sults in nucleic acid fragments wnose repetitive se~uences are blocked by complementary fragments such that sufficient unique sequence regions remain free for attacnment to chromosomal DNA during the in situ hybriCization step.

llI. Labeling the Nucleic Acid Fragments of tne Hetero~eneous Mixture.
-Several standard tecnniques are available forla~e'ing single and dou~le stranded nucleic acid fragments of the heterogeneous mixture. Tney incluae incorporation ~, ~ t~

of radioacti~e la~els, e.g. Harper et al. Chromosoma, Vol 8'1, pgs. 431-439 (1984); direct attacnment of fluorescent labe's, e.g. Smitn et al., Nucleic Aci~s Research, Vol. 13, pgs. 2399-2412 (1985), and Connolly et al., Nucleic Acids S Research, Vol. 13, pgs. 4485-45~2 (1985); and various chemical modifications of the nucleic acid fragments that render them detectable immunochemically or ~y other affin-ity reactions, e.g. Tchen et al., "Chemically Modified Nucleic Acids as Immunoaetectable Probes in Hybridization Experiments," Proc. ~atl. Acad. Sci., Vol 81, pgs. 3466-3470 (1984); Ricnardson et al., "Biotin and Fluorescent Labeling of RNA Using T4 RNA Ligase," Nucleic Acids Research, Vol. 11, pgs. 6167-6184 (1983); Langer et al., "Enzymatic Synthesis of Biotin-Labeled Polynucleotides:
Novel Nucleic Acid Affinity ProDes," Proc. Natl. Acad.
Sci., Vol. 78, pgs. 6633-6637 (1981); Brigati et al., "Detection of Vival Genomes in Cultered Cells and Paraffin-Embedded Tissue Sections Using Biotin-La~eled Hybridization Probes," Virology, Vol. 126, pgs. 32-50 (1983); Broker et al., "Electron Microscopic Visualization of tRNA Genes with Ferritin-Avidin: Biotin Labels," Nucleic Acids Research, Vol. 5, pgs. 363-384 (1978); Bayer et al., "The Use of tne AvidinBiotin Complex as a Tool in Molecular Biology,"
Methods of Biochemical Analysis, Vol. 26, pgs. 1-45 (1980) and Kunlmann, Immunoenzyme Techniques in Cytochemistry (~einheim, Basel, 1984).

()5 -All of tne labeling techni~ues disclosed in the a~ove references may be preferred under particular circum-stances.
Several factors govern the choice ~f labeling means, including the effect~ of the label on the rate of hybridization and binding of the nucleic acid frag-ments to the cnromosomal DNA, tne accessi~ility of tne bound probe to labeling ~oieties applied after initial hybridization, tne nature and intensity of tne signal generated by the label~ the expense and ease in which the la~e' is applied, and tne like.
Tne term labeled nucleic acid fragment as used herein comprenends la~eling means which include chemica modification of nucleic acid fragment by substituting 1~ derivatized ~ases, by forming adducts, or tne like, which after hydridization render tne nucleic acid fragments de-tectable by immunocnemical stains or affinity labels~ such as biotin-avid labelin9 systems, N-acetoxy-N-2-acetyl-aminofluorene (AFF) labeling systems, or tne li~e.
For most applications, labeling means whicn generate fluorescent signals are preferred.

. '~ ' .

IV. ln Situ H~bridi~ation.
Application of the neterogeneous mixture of tne invention to chromosomes is accomplisned by standard in situ nybridization tecnni~ues. Several execellent guides to the technique are availa~le. e.g., Gall and Pardue, ~Nucleic Acid Hybridization in Cytological Preparations,"
Methods in Enzymology, Vol. 21, pgs. 470-480 (1981);
Henderson, "Cytological Hybridization to Mammalian Chromo-somes," lnternational Review of Cytology, Vol. 76, pgs.
1-46 (1982); and Angerer, et al., ~ln Situ Hybridization to Cellular RNAs," in Genetic Engineering: Principles and Methods, Setlow and Hollaender, Eds., Vol. 7, pgs. 43-65 (Plenum Press, New York, 1985).
~hree fActors influence tne staining sensitivity of a heterogeneous mixture of hybridization probes: (1) 1~ efficiency of hybridization (fraction of target DNA that can be hybri~ized by probe), (2) detection efficiency (i.e., the amount of visi~le signal tnat can be obtained from a gi~en amount of hybridization probe), and (3) level of noise produced by nonspecific binding of probe or com-ponents of tne detection system.
6enerally in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be examined. (2) prehybridization treatment ~J, '.
~:J

lbU~ --.

of tne biological structure to increase accessibility of target DNA, and to reduce nonspecific binding, (3) hybri-dization of the neterogeneous mixture of probe to tne DNA
¦ in tne biological structure or tissue; (4) postnybridi2a-tion wasnes to remove prODe not bound in specific hybrids, ~ and (S) detection of tne hybridized probes of the ~etero-! geneous mixture. ~ne reagents used in each of these steps and tneir conditions of use vary depending on tne particu-lar cnromosomes being stained.
The following comments are meant to serve as a guide for applying tne general steps listed a~ove. Some experimentation may be required to establish optimal staining conditions for particùlar applications.
I Fixatives include acid alcohol solutions, acid 1~ acetone solutions, Petrunkewitsch's reagen$, and various aldenydes SUCh as formaldehyde, paraformaldehyde~ glutar-aldenyde, or the like. Preferably, ethanol-acetic acid or methanol-acetic acid solutions in a~out 3:1 proportions are used to fix tne chromosomes. for cells or cnromosomes in suspension, the fixation procedure disclosed by ~rask, f et al., in Science, Vol. 230, pgs. 1401-1402 (19~5), is ¦ preferred. -Briefly, K2C03 and dimethylsuberimidate (DM,S) are added (from a Sx concentrated stock solution, mixea immediately before use) to a suspension containing OS

a~out 5 x lo6 nuclei/ml. Final K2C03 and D~S con-centrations are 20 mM and 3 mM, respectively. After 15 minutes at 25C, tne pH is ad3usted from 10.0 to 8.0 by the addition of SO microliters of 100 mM citric acid per milli'iter of suspension. Nuclei are washed once by cen-trifugation (3009, 10 minutes, 4C in 50 mM kCl, 5 m~, Hepes buffer~ at pH 9.0, and 10 mM MgS04).
Preferably, before application of the stain, chromosomes are treated with agents to remove proteins.
Such agents include enzymes or mild acids. Pronase or proteinase K are the preferred enzymes. Optimization of deproteinization requires a combination of protease con-centration and digestion time that maximize hybrization, but does not cause unacceptable loss of morphological detail. Optimum conditions vary according to chromosome types and method of fixation. Thus, for particular appli-cations, some experimentation may be re~uired to opti~ize protease treatment.
Proteins can also be removed by mild acid extrac-tion, e.g., 0.02-0.2 N HCl, followed by nign temperature (e.g., 70C) washes.
In some cases pretreatment witn RNase may be desira~le to remove residual RNA from the fixed chromo-somes. Such removal can be accomplisned by incubation of the fixed chromoso~es in 50-100 microgram/milliliter RNase in 2~ SSC (where SSC is a solution of 0.15M NaCL and 0.01~: sodium citrate) for a period of 1-2 hours at room temperature.
The step of hybridi~ing the probes of the netero-geneous mixture to the chromosomal DNA involves (1) dena-turing the chromosomal DNA so that pro~es can gain access to complementary single stranded regions, and (2) applying the heterogeneous mixture under conditions which allow the probes to anneal to complementary sites. Methods for denaturation include incubation in the presence of high pH, low pH, hign temperature, or organic solvents SuCh as formamide, tetraalkylammonium halides, or the like, at various combinations of concentration and temperature.
The preferred denaturing procedure is incubation for between about 1-10 minutes in formamide at a concentration between about 35-95 percent in 2X SSC and at a temperature between about 25-70 C. Determination of the optimal incu~ation time, concentration, and temperature witnin tnese ranges depends on several variables~ including tne method of fixation and chromosome type.
After the cnromosomal DNA is denatured, tne dena-turing agents are removed before application of the ~etero-geneous mixture. Where formamide and heat are the primary denaturing agents, removal is conveniently accomplished by plunsing tne substrate or vessel containing the denatured 1. 3t !l~()S

chromosomes into an ice batn, and/or by several wasnes by an ice-cold solvent, such as a 70X, 80g, 95X cold etnanol series.
The ambient physiochemical conditions of the chromosomal DNA at the time the heterogeneous mixture is applied is referred to herein as the hybridization condit-ions, or annealing conditions. Optimal hybridization con-ditions for particular applications depend on several factors, including salt concentration, incubation time of cnromosomes in tne heterogeneous mixture, and the concen-trations> complexities, and lengths of the probes making up the neterogeneous mixture. Roughly, the nybridization conditions must be sufficiently denaturing to minimize nonspecific binding and hybridizations with excessive numbers of base mismatches. On the other nand, the condi-tions cannot be so stringent as to reduce hybridizations below detectable levels or to require excessively long incu~ation times. Generally, the hybridization conditions are much less stringent than the conditions for denaturing the chromosomal DNA.
The concentrations of probes in tne heterogeneous mixture is an important variable. The concentrations must be hign enoug~ so that the respective cnromosomal binding sites are saturated in a reasonable time (e.g., within about 18 hours), yet concentrations higher than tnat just t:~S

neceSsary to achieve saturation should be avoided so that nonspecific binding is minimized. A preferred concentra-tion range of nucleic acid fragments in the heterogeneous mixture is between about 1-10 nanograms per kilobase of complexity per milliliter.
The fixed chromosomes can be treated in several ways either during or after the hybridization step to reduce nonspecific binding of probe DNA. Sucn treatments include adding a large concentration of nonprobe, or "carrier", DNA to tne heterogeneous mixture, using coa~ing solutions, such as Denhardt's solution (Biocnem. Biopnys.
Res. Commun., Vol. 23, pgs. 641-645 (1966), witn tne heterogeneous mixture, incubating for several minutes, e.g., 5-20, in denaturing solvents at a temperature 5-10C above the hybridization temperature, and in the case of RNA probes, mild treatment with single strand RNase (e.g., 5-10 micrograms per millileter RNase) in 2X
SSC at room temperature for 1 hour).

Y. Making and Using a Staining Reagent Specific to Human Cnromosome 21.
A. Isolation of Chromosome 21 and Construction of a Chromosome 21-Specific Library DNA fragments from human chromosome-specific libraries are available from the National LaPoratory Gene Library Project througn tne American Type Culture 6~

Co~lection (A~CC), Rockville~ ~D Chromosome 2i-speci-ic DNA fr~g~ents ~ere gene-~ted ~y tne procedure descriDed by ~uscoe et ~1 , in ~Construction of ~ifteen Human ~hromosome-specific DNA Li~raries from Flow-Pu-ified ~1 Cn-om~somes,~enetic~ Cell Genetics 43:79 - 86 .
(19B6) Briefly, ~ numan diploid fi~robl~st culture was establisned from newborn ~oreskin tissue Chromosomes of tne cells ~ere isol~ted by t~e MgS04 nethod for v~n den Engh et ~1 , Cytometery, Vol. S, pgs. 108-123 (1984), and stained witn tne f)vorescent d~es ~oecns~ 33258 ~nd C~romomycin A3 Chromsome 21 ~s purified on tne L~wrence ~ivermore Nation~l LaDor~tor~ ~ign speed sorter, described by Peters et ~1., cytometrY~ Vol. 6, pgs. 290-301 (198~) lS After sorting, ehromDsome contentr~tions were ~pproximatel~ 4 x tO5/ml ~herefore, prior to DNA ex-tr~ction, the cnromosomes (0 2 - 1.0 x 106) were concen-tr~ted by centrifugation ~t 40,000 x 9 ~or 30 ~inutes ~t 4C The pellet w~s tnen resuspended in 100 ~icroliters of DNA isol~tion buffer (lS ~M N~Cl, 10 oM EDTA, 10 m~, ~ris HCl pH 8 0) containing 0 5X SDS ~nd 100 ~icrograms/ml protein~se K After overnignt incub~tion ~t 37C, tne protejns ~ere extrected twice with pncnol:cnloroform:
isoamyl elco~ol (25:24~ nd once witn cnloroform isoamyl ~lconol (24:~). Because of the small ~mounts of D~A, eacn s ,-l)S

organic phase was reextracted witn a small amount of 10 mM
Tris pH 8.0, 1 mM EDTA (TE). Aqueous layers were com~ined and transferred to a Schleicher and Scnuell mini-collodion membrane (#UH020/25) and dialyzed at room temperature against TE for 6-8 hours. ~ne purified DNA solution was tnen digested with 50 units of Hind III (Betnesda Research Laboratories, Inc.) in 50 mM NaCl, 10 mM Tris HCl pH 7.5, 10 m~: MgC12, 1 mM dithiothreitol. After 4 hours at 37, the reaction was stopped ~y extractions with pnenol and cnloroform as described above. Tne aqueous phase was dialyzed against water overnight at 4C in a mini-collodion bag and tnen 2 micrograms of Charon 21A arms cleaved with Hind III and treated with calf al~aline phos-phatase (Boehringer Mannheim) were added. Tnis solution was concentrated under vacuum to a volume of S0-100 micro-liters and transferred to a 0.5 ml microfuge tube where the DNA was precipitated with one-tenth volume 3M sodium acetate pH 5.0 and 2 volumes ethanol. The precipitate was collected by centrifugation, washed with cold 70X etnanol, and dissolved in 10 microliters of TE.
After allowing several hours for the DNA to ais-solve, 1 microliter of lOX ligase buffer (0.5M Tris HCl pH
7.4, 0.1 M MgC12, O.lM dithiothreitol, 10 mM ATP, 1 mg/ml bovine serum al~umtn) and 1 unit of T4 ligase (Betnesda Research Laboratory, Inc.) were added. The ligation reaC

tion was incubated at 10C for 1~-20 hours and 3 micro-liters aliquots were packaged into phage particles using in vitro extracts prepared from E. coli strains BH~ 2688 -and BHB 2690, described by Hohn in Methods in Enzymology, Vol. 68, pgs. 299-309 (1979) Molecular Cloning. A Labora-tory Manual, (Cold Spring Harbor Laboratory, New York, 1982). Briefly, botn extracts were prepared by sonication and combined at tne time of in vivo packaging. Tnese extracts packaged wild-type lambda DNA at an efficiency of 1-5 x 108 plaque forming units (pfu) per microgram. Tne resultant phage were amplified on E. coli LE392 at a den-sity of approximately 104 pfu/150 mm dish for 8 hours to prevent plaques from growing together and to minimize dif-ferences in growth rates of different recombinants. Tne phage were eluted from the agar in 10 ml SM buffer (50 mM
Tris HCl pH 7.5, 10 mM MgS04, 100 mM NaCl, O.OlX gelatin) per plate by gentle shaking at 4C for 12 hours. The plates were tnen rinsed with an additional 4 ml of SM.
After pelleting cellular debris, tne phage suspension was stored over cnloroform at 4C.
B. Construction and Use of Chromsome 21-Specific Stain for Staining Chromosome 21 of Human Lympnocytes Clones llaving unique sequence inserts are isolated ~y the method of Benton and Davis, Science, Vol. 196, pgs.
180-182 (1977~. Briefly, about 1000 recombinant phage are s - 3~ -isolated at random from the chromosome 21-specific li~rary.
These are transferred to nitrocellulose and probed witn nick translated total genomic human DNA.
0f the clones which do not ShOW strong hybriai2a-tion, approximately 300 are picked whicn contain apparent uni~ue se~uence DNA. After the selected clones are ampli-fied, the chromosome 21 insert in each clone is 32p la~eled and hybridized to Soutnern nlots of numan genomic DNh di-gested with the same enzyme used to construct the cnromo-some 21 library, i.e., Hind 111. Unique se~uence contain-ing clones are recognized as those that produce a sing7e band during Southern analysis. Roughly~ 100 such clones are selected for the heterogeneous mixture. The unique sequence clones are amplified, the inserts are remove~ by Hind III digestions, and the inserts are separated from the p~age arms by gel electrophoresis. The probe DNA
fragments (i.e., the unique sequence inserts) are removed from the gel and biotinylated by nic~ translation (e.g., by a kit available from Betnesda Research Laboratories).
La~eled DNA fragments are separated from tne nick transla-tion reaction using small spin columns made in 0.5 m' ~B eppendorph tubes filled witn Sepnadex G-50 (medium) swollen in 50 mM Tris, 1 mM EDTA, 0.1X SDS, at pH 7.5.
Human lymphocyte chromosomes are prepared fo!lowing Harper et al, Proc. Natl. Acad. Sci., Vol. 78, pgs. 4458-4460 (1981). Metaphase and interphase cells were washed 3 ~ ~r~Je ~Q~

()S

times in pnosphate buffered saline, fixed in methanol-acetic acid (3:1) and dropped onto cleaned microscope slides. Slides are stored in a nitrogen atmosphere at -20C.
Slides carrying interphase cells and/or metaphase spreads are removed from the nitrogen, neated to 65 C
for 4 hours in air, treated with RNase (lO0 micrograms/ml for l hour at 37C), and denydrated in an etnanol series.
They are then treated with proteinase K (60 ng/ml at 37C
for 7.5 minutes) and dehydrated. The proteinase K concen-tration is adjusted depending on the cell type and enzyme lot so that almost no phase microscopic image of the chro-mosomes remains on the dry slide. The hybridization mix consists of (final concentrations) 50 percent formamide, 2X SSC, 10 percent dextran sulfate, 500 micrograms/ml carrier DNA (sonicated herring sperm DNA), and 2.0 micro-gram/ml biotin-labeled chromsome 21-specific D~A. Tnis mixture is applied to the slides at a density of 3 micro-liters/ cm2 under a glass coverslip and sealed witn rub~er cement. After overnignt incu~ation at 37C, tne slides are washed at 45C (50X formamide-2XSS pH 7, 3 times 3 minutes; followed by 2XSSC pH 7, 5 times 2 minutes) and immersed in BN buffer (0.1 M Na bicarbonate, 0.05 percent NP-40, pH 8). The slides are never allowed to dry after t~is point.

- ~8 -Tne 51 ides are removed from the 8N ~uffer and b)ocked for 5 minutes at room temperature witn BN buffer containing SX non-fat dry milk (Carnation) and 0.~2~ Na Azide (5 microliter/cm under plastic coverslips). Tne S coverslips are removed, and excess li~uid briefly drained and fluorescein-avidin DCS (3 microgram/ml in BN buffer with 5X milk and 0.02X NaAzide) is applied (S microliter/
cm2). The same coverslips are replaced and tne slides incubated 20 minutes at 37C. The slides are t~en washed 3 times for 2 minutes each in BN buffer at 45C. Tne intensity of biotin-linked fluorescence is amplified Dy adding a layer of biotinylated goat anti-avidin anti~ody (S microgram/ml in Bl~ buffer with 5% goat serum and 0.02X
NaAzide), followed, after wasning as above, ~y anotner lS layer of fluorescein-avidin DCS. Fluorescein-avidin DCS, goat antiavidin and goat serum are all available commer-cially, e.g., Vector Laboratories (Burlingame, CA). After washing in BN, a fluorescence antifade solution, p-phenyl-enediamine (l.S microliter/cm of coverslip) is added before observation. It is important to keep thiS layer thin for optimum microscopic imaging. ThiS antifade significantly reduced fluorescein fading and allows continuous microscopic observation for up to 5 minutes.
The DNA counterstains (DAPI or propidium iodide) are included in the antifade at 0.25-0.5 microgram/ml.
/e~c~r~/~

The red-fluorescing DNA-specific dye propidium iodide (PI) is used to allow simultaneous observation of hybridized probe and total DNA. Tne fluorescein and PI
are excited at 450-490 nm (Zeiss filter combination 487709). Increasing the excitation wavelengtn to 546 nm (Zeiss filter combination 487715) allows observation of the PI only. DAPI, a blue fluorescent DNA-specific stain excited in the ultraviolet (Zeiss filter combination 487701), is used as the counterstain wnen biotin-la~eled and total DNA are observed separately. Metaphase chromo-some 21s are detected by randomly located spots of yellow distributed over the body of tne chromosome.

VI. Chromosome 21-Specific Staining Oy Blocking Repetitive Probe and Chromosomal DNA
Hign concentrations of unlabeled human genomic DNA and lambda phage DNA were used to inni~it the binding of repetitive and vector DNA sequences to the target ChromoSomeS. Heavy protejnase digestion and sunsequent fixation improved access of probes to target DNA.
Human metapnase spreads were prepared on microscope slides with standard techniques and stored 2~ immediately in a nitrogen atmospnere at -20C.
Slides were removed from tne freezer and allowed to warm to room temperature in a nitrogen atmosphere before beginning the staining procedure. Tne warmed slides were first treated with 0.6 microgram/ml proteinase K in P buffer (2U mM Tris, 2 mM CaC12 at pH
7,5) for 7.5 minutes, and washed once in P buffer. The arnount of proteinase K used needs to be adjusted for tne particular enzyme lot and cell type. Next the slides were wasned once in paraformaldenyde buffer (pnosphate buffered saline (PBS) plus 50 mM MgC12, at pH 7.5), immersed for 10 minutes in 4% paraformaldenyde in paraformaldehyde buffer, and wasned once in 2XSSC (0.3 M NaCl, 0.03 M
sodium citrate at pH 7). DNA on the slides was denatured by immersing in 70X formamide and 2XSSC at 70C for 2 minutes. After denaturing the slides were stored in 2XSSC. A hybridization mix was prepared which consisted B of 50X formamide, 10~ dextran sulfate, lX Tween 20, 2XSSC, 0.5 mg/ml human genomic DNA, 0.03 mg/ml lambda DNA, and 3 microgram/ml biotin labeled probe DNA. The probe DNA
consisted of the nignest density fraction of pna9e from the chromosome 21 Hind III fragment library (ATCC
accession number 57713), as determined ~y a cesium cnloride gradient. (Botn insert and pnage DNA of the probe were labeled by nick translation.) The average insert size (amount of chromosome 21 DNA), as determined by gel electrophoresis is about 5 kilobases. No attempt was made to remove repetitive sequences from tne inserts or to isolate the inserts from tne lam~da phage vector.
~k f~a ~ /`k VS

The hybridization mix was denatured by heating to 70C for five minutes followed by incubation at 37C for one hour.
The incubation allows the human genomic DNA and unlabelled lambda DNA in the hybridization mix to block the human repetitive sequences and vector sequences in the probe.
The slide containing the human metaphase spread was removed from the 2XSSC and blotted dry with lens paper.
The hybridization mix was immediately applied to the slide, a glass cover slip was placed on the slide with rubber cement, and the slide was incubated overnight at 37C.
Afterwards, preparation of the slides proceeded as described in Section V (wherein chromosome 21 DNA was stained with fluorescein and total chromosomal DNA counterstained with DAPI).
The descriptions of the foregoing embodiments of the invention have been presented for purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with ,~

V~

-- ~2 various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.

Claims (17)

1. A chromosome-specific staining reagent com-prising a heterogeneous mixture of labeled nucleic acid fragments, the labeled nucleic acid fragments being derived from the same chromosome type, and being substantially free of repetitive nucleic acid sequences having hybridization capacity.

2. The chromosome-specific staining reagent according to claim 1 wherein said chromosome type is an abnormal human chromosome type.

3. The chromosome-specific staining reagent according to claim 1 wherein said chromosome type is selected from the group consisting of normal human chromo-somes 1 through 22, X, and Y.

4. The chromosome-specific staining reagent of claim 3 wherein said heterogeneous mixture comprises labeled nucleic acid fragments derived from substantially equal amounts of between about 10-1000 distinct cloned inserts.

5. The chromosome-specific staining reagent of claim 4 wherein said labeled nucleic acid fragments are derived from substantially equal amounts of between about 10-1000 distinct cloned inserts each having a length within the range of between about 20-45 kilobases.

6. The chromosome-specific staining reagent of claim 5 wherein said distinct cloned inserts form said labeled nucleic acid fragments, each fragment having a length within the range of between about 50-500 bases.

7. The chromosome-specific staining reagent of claim 6 wherein said labeled nucleic acid fragments are single stranded.

8. The chronosome-specific staining reagent of claim 7 wherein said labeled nucleic acid fragments are biotinylated or modified with N-acetoxy-N-2-acetylamino-fluorene.

9. The chromosome-specific staining reagent of claim 4 wherein said labeled nucleic acid fragments are derived from substantially equal amounts of between about 100-400 distinct cloned unique sequence inserts.

10. The chromosome-specific staining reagent of claim 9 wherein said distinct cloned inserts form said labeled nucleic acid fragments, each fragment having a length within the range of between about 50-5000 bases.

11. The chromosome-specific staining reagent of claim 10 wherein said distinct cloned inserts form said labeled nucleic acid fragments, each fragment having a length within the range of between about 50-500 bases.

12. The chromosome-specific staining reagent of claim 11 wherein said labeled nucleic acid fragments are single stranded.

13. The chromosome-specific staining reagent of claim 12 wherein said labeled nucleic acid fragments are biotinylated or modified with N-acetoxy-N-2-acetylamino-fluorene.

14. A chromosome-specific staining reagent produced by the process of:
isolating chromosome-specific DNA;
cloning pieces of the isolated chromosome-specific DNA;
disabling the hybridization capacity of repeated sequences contained in the cloned pieces of the isolated chromosome-specific DNA to form a collection of nucleic acid fragments which hybridize to unique chromosomal DNA
sequences;
labeling the nucleic acid fragments of the collection to form a heterogeneous mixture of nucleic acid fragments.

15. The chromosome-specific staining reagent of claim 14, wherein said step of disabling said hybridization capacity includes selecting cloned pieces of said isolated chromosome-specific DNA which nave substantially unique base sequences.

16. The chromosome-specific staining reagent of claim 14 wherein said step of disabling said hybridization capacity includes prereassociating said cloned pieces of said isolated chromosome-specific DNA With unlabeled repetitive sequence nucleic acid fragments.

17. A method of staining chromosomal DNA of a particular chromosome type or portion thereof, or a particular group of chromosome types, the method comprising the steps of:
providing a heterogeneous mixture of labeled nucleic acid fragments, substantial portions of each labeled nucleic acid fragment in the heterogeneous mixture having base sequences substantially complementary to base sequences of the chromosomal DNA; and reacting the heterogeneous mixture with the chromosomal DNA by in situ hybridization.

(18) A chromosome-specific staining reagent comprising a heterogeneous mixture of labeled nucleic acid fragments which is substantially free of repetitive nucleic acid sequences having hybridization capacity, and which can be used to stain specific chromosomes, specific subsets of chromosomes, or specific subregions of specific chromosomes whether the targeted sequences are present at genomically normal or abnormal copy numbers.
(19) The chromosome-specific staining reagent according to claim 18 wherein said labeled nucleic acid fragments are derived from an abnormal human chromosome type.
(20) The chromosome-specific staining reagent according to claim 18 wherein said labeled nucleic acid fragments are derived from the group consisting of normal human chromosomes 1 through 22, X, and Y.
(21) The chromosome-specific staining reagent of claim 18 wherein said labeled nucleic acid fragments are single stranded.
(22) The chromosome-specific staining reagent of claim 21 wherein said labeled nucleic acid fragments are biotinylated or modified with N-acetoxy-N-2-acetylamino-fluorene.
(23) A chromosome-specific staining reagent, which can be used to stain specific chromosomes, specific subsets of chromosomes, or specific subregions of specific chromosomes whether the targeted sequences are present at genomically normal or abnormal copy numbers, produced by the process of:
isolating chromosome-specific DNA;
cloning pieces of the isolated chromosomes-specific DNA;
disabling the hybridization capacity of repeated sequences contained in the cloned pieces of the isolated chromosome-specific DNA; and labeling the nucleic acid fragments of the collection to form a heterogeneous mixture of nucleic acid fragments.
(24) The chromosome-specific staining reagent of claim 23, wherein said step of disabling said hybridization capacity includes selecting cloned pieces of said isolated chromosome-specific DNA which have substantially unique base sequences.
(25) The chromosome-specific staining reagent of claim 23 wherein said step of disabling said hybridization capacity includes prereassociating said cloned pieces of said isolated chromosome-specific DNA with unlabeled repetitive sequence nucleic acid fragments.
(26) A chromosome-specific staining reagent, which can be used to stain specific chromosomes, specific subsets of chromosomes, or specific subregions of specific chromosomes whether the targeted sequences are present at genomically normal or abnormal copy numbers, produced by the process of:
isolating chromosome-specific DNA;
cloning pieces of the isolated chromosome-specific DNA;
generating RNA transcripts from the cloned pieces of said isolated chromosome-specific DNA;
disabling the hybridization capacity of repeated sequences contained in the RNA transcripts; and labeling the RNA trancripts either during or after their generation to form a heterogeneous mixture of labeled RNA fragments.
(27) A chromosome-specific staining reagent, which can be used to stain specific chromosomes, specific subsets of chromosomes, or specific subregions of specific chromosomes whether the targeted sequences are present at genomically normal or abnormal copy numbers, produced by the process of:

isolating chromosome-specific DNA;
cloning pieces of the isolated chromosome-specific DNA;
disabling the hybridization capacity of repeated sequences contained in the cloned pieces of the isolated chromosome-specific DNA;
generating RNA transcripts from said nucleic acid fragments; and labeling the RNA transcripts either during or after their generation to form a heterogeneous mixture of labeled RNA fragments.
(28) A method of staining chromosomal DNA of a particular chromosome type or portion thereof, or a particular group of chromosome types or portions thereof, the method comprising the steps of:
providing a heterogeneous mixture of labeled nucleic acid fragments, substantial portions of each labeled nucleic acid fragment in the heterogeneous mixture having base sequences substantially complementary to base sequences of the chromosomal DNA; and reacting the heterogeneous mixture with the chromosomal DNA by in situ hybridization; wherein said method can be used to stain said chromosome type or portion thereof or said group of chromosome types or portions thereof whether the targeted sequences are present at genomically normal or abnormal copy numbers.
(29) The method of staining chromosomal DNA according to claim 28, wherein said chromosomal DNA is human chromosomal DNA.
(30) The method of staining chromosomal DNA according to claim 28, wherein said heterogeneous mixture of labeled nucleic acid fragments are DNA fragments or RNA fragments.
(31) The method of staining chromosomal DNA according to claim 30 wherein said nucleic acid fragments are selected or generated from the group consisting of total chromosome-derived recombinant library DNA, DNA inserts purified from a chromosome-derived recombinant library, and specific DNA
fragments derived from said recombinant library.
(32) The method according to claim 28, wherein the heterogeneous mixture of labeled nucleic aicd fragments are selected from the group consisting of nucleic acid fragments labeled with radioactivity, nucleic acid fragments labeled with a fluorescent label, nucleic acid fragments labeled with an enzyme and nucleic acid fragments labeled with at least one member of a specific binding pair.
(33) A method of detecting chromosomal abnormalities in human cells, said method comprising the steps of:
(a) denaturing a hybridization mix comprising unlabeled human genomic DNA, unlabeled nonhuman genomic DNA
and a heterogeneous mixture of labeled nucleic acid fragments;
(b) incubating said denatured hybridization mix to block labeled repetitive sequences;
(c) combining a denatured DNA target sample with said denatured hybridization mix under conditions appropriate for hybridization of complementary nucleic acid sequences to occur; and (d) detecting labeled human nucleic acid in said target sample;
wherein said method can detect target sequences whether present at genomically normal or abnormal levels of abundance.
(34) A method according to claim 33 wherein said unlabeled nonhuman DNA is herring sperm DNA.
(35) The method according to claim 33 wherein said chromosomal abnormalities are selected from the group consisting of extra or missing individual chromosomes, extra or missing portions of chromosomes, and chromosomal rearrangements.
(36) The method according to claim 33 wherein said heterogeneous mixture of labeled nucleic acid fragments is selected or generated from a group consisting of total chromosome-derived recombinant library DNA, DNA inserts purified from a chromosome-derived recombinant library and specific fragments derived from said recombinant library and/
or from a group consisting of corresponding DNA containing analogous base sequences to the DNA sequences of said first group.
(37) The method according to claim 33 wherein said heterogeneous mixture of labeled nucleic acid fragments are derived from the group consisting of human chromosomes 1 through 22, X, and Y.
(38) The method according to claim 33 wherein said heterogeneous mixture of labeled nucleic acid fragments are selected from the group consisting of nucleic acid fragments labeled with radioactivity, nucleic acid fragments labeled with a fluorescent marker, nucleic acid fragments labeled with an enzyme and nucleic acid fragments labeled with at least one member of a specific binding pair.
(39) The method according to claim 35, wherein said chromosomal abnormalities are selected from the group consisting of abnormalities of human chromosomes 1 through 22, X, and Y.
(40) A method of detecting chromosome abnormalities in human cells, said method comprising the steps of:
(a) combining (1) human cells treated so as to render nucleic acid sequences present available for hybridization with complementary nucleic acid sequences; and (2) a hybridization mixture comprising a heterogeneous mixture of a labeled nucleic acid fragments derived from a specific chromosome; unlabeled human genomic DNA, and unlabeled nonhuman genomic DNA under conditions appropriate for hybridization of complementary nucleic acid sequences to occur; and (b) detecting labeled human nucleic acid fragments hybridized to nucleic acid sequences from human cells wherein said method can detect nucleic acid sequences in the cells whether present at genomically normal or abnormal levels of abundance.
(41) A method according to claim 40 wherein said unlabeled nonhuman genomic DNA is herring sperm DNA.
(42) The method according to claim 40 wherein said chromosomal abnormalities are selected from the group consisting of extra or missing individual chromosomes, extra or missing portions of chromosomes, and chromosomal rearrangements.
(43) The method according to claim 42, wherein said chromosomal abnormalities are selected from the group consisting of abnormalities of human chromosomes 1 through 22, X and Y.
(44) The method according to claim 42 wherein said abnormality is aneuploidy and said heterogeneous mixture of labeled nucleic acid fragments derived from the selected chromosome comprises clones from a chromosome-specific recombinant DNA library.
(45) The method according to claim 44 wherein the selected chromosome is chromosome 21.
(46) The method according to claim 42 wherein said abnormality is aneuploidy and said heterogeneous mixture of labeled nucleic acid fragments derived from the selected chromosome comprises DNA inserts purified from clones in a chromosome-specific recombinant DNA library.
(47) The method according to claim 46 wherein the selected chromosome is chromosome 21.
(48) The method according to claim 44 wherein said heterogeneous mixture of labeled nucleic acid fragments is derived from RNA transcripts generated from clones from a chromosome-specific recombinant DNA library.
(49) A method of detecting specific diseases associated with abnormalities in the amount or organization of specific DNA sequences comprising the steps of:
choosing a mixture of labeled nucleic fragments which is substantially free of repetitive nucleic acid sequences having hybridization capacity and which has the capacity to stain the appropriate specific chromosomes, specific subsets of chromosomes, or specific subregions of specific chromosomes associated with the specific disease whether the sequences targeted are present at genomically normal or abnormal copy numbers;
reacting the heterogeneous mixture with the chromosomal DNA to be tested by in situ hybridization; and identifying the stained portions of the chromosomal DNA to determine whether abnormalites in the amount or organization of specific DNA sequences are present.
(50) A method according to claim 49 wherein the disease is Down's syndrome and the heterogeneous mixture of labeled nucleic acid fragments is derived from a chromosome 21-specific recombinant library.
(51) A method according to claim 49 wherein the disease is chronic myelogeneous leukemia and the heterogeneous mixture of labeled nucleic acid fragments contain sequences derived from chromosome 9 and chromosome 22.
(52) A chromosome-specific staining reagent according to claim 18 wherein the nucleic acid fragments were propagated in one or more vectors.
(53) A chromosome-specific staining reagent according to claim 52 wherein the one or more vectors is or are selected from the group consisting of cosmids, bacteriophages, and plasmids.