Proc. Nati. Acad. Sci. USAVol. 85, pp. 9138-9142, December 1988Genetics

Fluorescence in situ hybridization with human chromosome-specificlibraries: Detection of trisomy 21 and translocations ofchromosome 4

(tumor cytogenetics/prenatal diagnosis/aneuploidy/interphase)

D. PINKEL*, J. LANDEGENTt, C. COLLINS, J. FUSCOEt, R. SEGRAVES, J. LUCAS, AND J. GRAYBiomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550

Communicated by Robert T. Schimke, July 25, 1988

ABSTRACT Chromosomes can be specifically stained inmetaphase spreads and interphase nuclei by in situ hybridiza-tion with entire chromosome-specific DNA libraries. Unlabeledhuman genomic DNA is used to inhibit the hybridization ofsequences in the library that bind to multiple chromosomes.The target chromosome can be made at least 20 times brighterper unit length than the others. Trisomy 21 and translocationsinvolving chromosome 4 can be detected in metaphase spreadsand interphase nuclei by using this technique.

The application of nonradioactive in situ hybridization withchromosome-specific probes for cytogenetic analysis hasincreased significantly in recent years. This has been due tothe technical convenience of these procedures (1-3) and tothe increased availability of repetitive probes that hybridizeintensely and specifically to selected chromosomes. Suchprobes are now available for over half of the human chro-mosomes. In general, these bind to repeated sequences oncompact regions of the target chromosome near the centro-mere. However, one probe has been reported that hybridizesto human chromosome 1p36, and there are several probesthat hybridize to human chromosome Yq (ref. 4 and citationstherein). Hybridization with these probes permits rapididentification of chromosomes in metaphase spreads, deter-mination of the number of copies of selected chromosomes ininterphase nuclei (5-7), and determination of the relativepositions of chromosomes in interphase nuclei (4-6, 8-11).Interphase analysis is especially promising in clinical appli-cations where rapid results are desirable (e.g., in prenataldiagnosis), where cells are difficult to culture in vitro, orwhere concern exists about the ability to stimulate a repre-sentative fraction of cells into proliferation for metaphaseanalysis (e.g., in tumor studies). However, many applicationsare still limited by the lack of appropriate probes. For ex-ample, probes with sufficient specificity for prenatal diagnosisare not available for chromosome 13 or 21. In addition,repetitive probes are not very useful for detection of structuralaberrations since the probability is low that the aberrationswill involve the region to which the probe hybridizes.The development of procedures to construct high intensity

probes of any desired specificity would advance a broadspectrum of cytogenetic studies. One approach is to usecomposite probes made from numerous sequences clonedfrom the desired target region. Recombinant DNA librariesfor each of the human chromosomes, available from theAmerican Type Culture Collection, have been constructed bythe National Laboratory Gene Library Project (12). Thesecan be used as starting material for chromosome-specificcomposite probes. The principal difficulty of using suchlibraries is that they contain chromosome-specific sequences

and sequences shared with other chromosomes. Therefore,the chromosome-specific sequences must be selected orhybridization of the nonspecific sequences must be blocked(13). We report here that competitive hybridization usingentire chromosome-specific libraries for chromosomes 4 and21 as probes and human genomic DNA as the competitorallows intense and specific fluorescent staining of humanchromosomes 4 and 21 in metaphase spreads and interphasenuclei. We call this general approach "chromosome paint-ing." Translocations and aneuploidy involving these chro-mosomes can be strikingly visualized. We also present dataon hybridization with collections of unique sequence probeson chromosome 4.

MATERIALS AND METHODSCells. Metaphase spreads from human lymphocytes were

prepared from methotrexate-synchronized cultures by usingthe procedure of Harper et al. (14). These and all other cellswere fixed in methanol/acetic acid, 3:1. Other human lym-phocyte cultures were irradiated with 6Co y-rays and stim-ulated with phytohemagglutinin. Colcemid was added 48 hrafter stimulation and metaphase spreads were prepared 4 hrlater. Metaphase spreads and interphase cells from lympho-blastoid cells (GM03716A; Human Mutant Cell Repository,Camden, NJ) carrying trisomy 21 were prepared after a 4-hrcolcemid block. Interphase cells from the cell line RS4;11carrying t(4;11) and isochromosome 7q were harvested, fixedin methanol/acetic acid, and dropped onto slides (15). Slideswere stored at -20°C in plastic bags filled with nitrogen gas.

Probes. Human chromosome-specific libraries producedby the National Laboratory Gene Library Project (12) werethe source of all probes.Chromosome 4 unique sequences. One hundred and

twenty clones carrying chromosome 4-specific unique se-quence inserts selected from the Charon 21A libraryLL04NS01 were supplied by C. Gilliam (Harvard University)(16). The human inserts were all =3 kilobases (kb) in length,so the ratio of insert to vector DNA was <0.1. Total phageDNA was produced from each clone individually usingDEAE-cellulose columns (Whatman DE-52) (17). DNApooled from 3 or all 120 clones was biotinylated by nick-translation with biotin-11-dUTP (Bethesda Research Labo-ratories) and recovered at a concentration of ==20 ng/,ul usingSephadex G-50 spin columns.pBS-4. The entire chromosome 4 library LL04NS02 was

subcloned into Bluescribe plasmids (Stratagene, La Jolla,CA) to form the library pBS-4. The average insert to vector

*To whom reprint requests should be addressed.tPermanent address: Department of Experimental Hematology,Leiden University Medical Center, P.O. Box 9600, Leiden, TheNetherlands.tPresent address: Center for Environmental Health, U-39, Univer-sity of Connecticut, Storrs, CT 02628.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 85 (1988) 9139

DNA ratio in pBS-4 is =1. The plasmid library was amplifiedin bulk and the DNA was extracted and biotinylated asdescribed above. In some experiments, the biotinylated DNAwas concentrated by ethanol precipitation to achieve higherprobe concentrations.pBS-21. The entire chromosome 21 library LL21NS02 was

subcloned into Bluescribe plasmids to form the librarypBS-21. This library was amplified and biotinylated as de-scribed above.Human genomic DNA. Placental DNA (Sigma) was

treated with proteinase K, extracted with phenol, and soni-cated to a size range of 200-600 base pairs (bp).In Situ Hybridization. Unique sequence hybridization.

Hybridization was accomplished by using a modification ofthe procedure described by Pinkel et al. (5). The slide-mounted cells were treated with RNase (100 ,ug/ml in 0.3 MNaCl/30 mM sodium citrate at 37TC for 1 hr), dehydrated ina 70%/85%/100% ethanol series, treated with proteinase K(0.3-0.6 gg/ml in 20 mM Tris/2 mM CaCl2, pH 7.5, for 7.5min at 370C), and fixed [4% paraformaldehyde in phosphate-buffered saline (PBS; in g/liter, KCI, 0.2; KH2PO4, 0.2;NaCl, 8; Na2HPO4 7H2O, 2.16) plus 50 mM MgCl2 for 10 minat room temperature]. The DNA in the target cells wasdenatured by immersion in 70%o formamide/0.3 M NaCl/30mM sodium citrate, pH 7, for 2 min at 70°C. The hybridizationmixture [10 ,l total volume consisting of 50%o formamide, 0.3M NaCl/30 mM sodium citrate (final concentration), 10%dextran sulfate, 50 ,g of sonicated herring DNA per ml, and3-6 ng of biotinylated chromosome 4 unique sequences (40-80 ng of total phage DNA)] was then denatured (70°C for 5min) and applied. Hybridization was at 37°C overnight (16hr). Slides were washed in three changes of50o formamide/0.3 M NaCl/30 mM sodium citrate (final concentration), pH7, at 45°C for 5 min each and once in PN buffer (a mixture of0.1 M NaH2PO4 and 0.1 M Na2HPO4 to give pH 8/0.1%Nonidet P-40). The slides were then treated with alternatinglayers of fluoresceinated avidin and biotinylated goat anti-avidin, both at 5 ,g/ml in PNM buffer (PN buffer/5% non-fatdry milk/0.02% sodium azide, centrifuged to remove solids),for 20 min each at room temperature until three layers ofavidin were applied. The avidin and goat anti-avidin treat-ments were separated by three washes of 3 min each in PNbuffer [avidin (DCS grade) and anti-avidin from VectorLaboratories (Burlingame, CA)]. After the final avidin treat-ment, a fluorescence antifade solution (18) containing 1 ,ug of4',6-amidino-2-phenylindole or propidium iodide per ml wasapplied to stain double-stranded DNA (1.5 /l/cm2 under ano. 1 coverslip).Whole Library Hybridization. Hybridization was as above

except that RNase, proteinase K, and paraformaldehydewere not used. The amount ofprobe and genomicDNA in thehybridization mixture and the length of the hybridizationvaried as described in Results. All probe concentrations referto the human insert DNA unless otherwise noted. DNAconcentrations were determined by fluorometric analysis(Hoeffer Scientific Instruments, San Francisco). Incubationof hybridization mixture prior to hybridization followed twodifferent protocols. Protocol I. The hybridization mixture (10,ul) contained 10-150 ng of biotinylated human DNA (20-300ng of total plasmid DNA) and 0-10 ,g of unlabeled genomicDNA. The mixture was heated to denature the DNA andincubated at 37°C for a time t before it was added to the slide.Hybridization times ranged from 2 to 110 hr. Protocol II. Thiswas identical to protocol I except that an additional aliquot offreshly denatured genomic DNA was added to the hybrid-ization mixture after an incubation time t. The mixture wasthen incubated an additional time t prior to starting thehybridization. The volume of the hybridization mixture wasincreased <20%o by the additional genomic DNA.

Microscopy. Quantitative fluorescence measurementswere performed using a video camera on the microscope anda digital image processing system (4). The combination of redpropidium iodide and green-yellow fluorescein isothiocya-nate fluorescence appears as yellow in the photographs.

RESULTSWhole Library Hybridization. Fig. la shows hybridization

of pBS-4 to a human metaphase spread with a probe con-centration of 1 ng/,ul. No genomic DNA was used and thehybridization mixture was applied immediately after dena-turation. All chromosomes are stained, except near manycentromeres, with the two copies of chromosome 4 beingstained most heavily. The inclusion of unlabeled total humanDNA in the hybridization mixture substantially increased thespecificity of hybridization to chromosome 4. Fig. 1 b and cshow the result of a protocol I hybridization [0.8 ng of probeper /tl and 24 ng of genomic DNA per dul (Q = 2, seeDiscussion); 1-hr probe incubation and 110-hr hybridization].This probe concentration corresponds to =0.5 x 10-2 pg/tklper kilobase of unique sequence, assuming that 30% of thechromosomal DNA is unique. Quantitative image analysisshows that the intensity per unit length of chromosome 4 is-20 times that of the other chromosomes. This is referred toas the contrast ratio. Two layers of avidin-fluorescein iso-thiocyanate have been used here to make the non-targetchromosomes sufficiently bright to be measured accurately.However, the number 4 chromosomes can be recognizedeasily after a single layer.

Fig. ld demonstrates detection of a radiation-inducedtranslocation involving chromosome 4 in human lympho-cytes [protocol I, 1 ng of probe per /ul and 76 ng of genomicDNA per /ul (Q = 5, see Discussion); 1-hr probe incubationand 16-hr hybridization]. The contrast ratio was -5. Fig. leshows that the normal and two derivative chromosomesresulting from the t(4;11) in cell line RS4;11 can be detectedin interphase nuclei as three distinct domains [protocol I, 13.5ng of probe per ,ul and 800 ng of genomic DNA per ul (Q =5, see Discussion); 1-hr probe incubation and 16-hr hybrid-ization]. The increased probe concentration resulted inbrighter signals relative to Fig. id. Approximately half of thecells clearly show the presence of three nuclear domains.

Hybridization of pBS-21 to a metaphase spread from a cellline with trisomy 21 is shown in Fig. lf [protocol II, 4 ng ofprobe per /4 and 250 ng of genomic DNA per /l; 3-hrincubation, additional 250 ng of genomic DNA per /ul (Q = 1+ 1, see Discussion); 3-hr probe incubation and 16-hrhybridization]. A small amount of hybridization is visiblenear the centromeres of the other acrocentrics. Fig. lg showstwo interphase nuclei from this same hybridization. Theseclearly show the three chromosome 21 domains. Hybridiza-tion with probe prepared according to protocol I resulted inhigher relative intensity of the shared signals on the D- andG-group chromosomes, and consequently it was more diffi-cult to determine the number of number 21 chromosomes ininterphase (not shown). Increasing stringency by using ahybridization mixture with 55% formamide and 0.15 MNaCl/15 mM sodium citrate, which lowers the meltingtemperature about 8°C, did not reduce the unwanted hybrid-ization. Addition of unlabeled pA ribosomal DNA (19) alsowas ineffective at increasing specificity.Unique Sequence Hybridization. Hybridization with a pool

of three chromosome 4 unique sequences was conducted ata total phage concentration of 2 ng//l per clone, correspond-ing to 50 pg//l per kilobase of human insert. Individualhybridization sites could be located to within a fraction of thewidth of a chromatid (Fig. lh) after overnight hybridization(16 hr) and application of three layers of avidin. Analysis of45 spreads in two experiments showed 265 fluorescent spots

Genetics: Pinkel et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

I~~~~II

FIG. 1. (a) Hybridization of pBS-4 DNA with no competing genomic DNA to a human metaphase spread. Both copies of chromosome 4,which are brighter than the others, are indicated by large arrows. Small arrows indicate regions that are unstained with the probe. (b)Hybridization of pBS-4 to a human metaphase spread with Q = 2 of genomic DNA. Chromosome 4 is yellow; others are red due to the propidiumiodide counterstain. (c) Same spread as b but using a filter that passes only the fluorescein isothiocyanate fluorescence. (d) Detection of aradiation-indcei-pd transloca.tion (aqrrows,) involving chromosome 4 in a human metaphase spread. (e) Representative nuclei from cell line RS4;11that homogeneously contain t(4;11). (f) Hybridization of pBS-21 to a metaphase spread of a trisomy 21 cell line. (g) As infbut interphase nuclei.(h) Hybridization of three unique sequence probes from chromosome 4 to a human metaphase spread.

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Proc. Natl. Acad. Sci. USA 85 (1988) 9141

on chromosome 4 out of the 540 possible (3 target sites perchromatid x 4 chromatids per metaphase x 45 metaphases),indicating a hybridization efficiency of -50%. The totalnumber of spots on all chromosomes was 568 (265 on chro-mosome 4 plus 303 on other chromosomes), giving a hybrid-ization specificity of -50%. Analysis of 3 spreads from a 16-hrhybridization with the complete collection of 120 uniquesequence probes at a total probe concentration the same as forthe group of 3 (resulting in -1.5 pgfl~d per kilobase of humaninsert) showed 222 fluorescent spots out of the 1440 possibleon the number 4 chromosomes (120 target sites per chromatidx 4 chromatids per metaphase x 3 metaphases). Thus thehybridization efficiency was 15%. Eight hundred fourteentotal spots were found on all of the chromosomes, giving aspecificity of27%. These experiments indicate that substantialhybridization can occur with unique sequence probes at lowprobe concentrations.

DISCUSSIONCompetitive Hybridization. This paper describes the use of

unlabeled genomic DNA to competitively inhibit hybridiza-tion of those sequences in a chromosome-specific library thatare shared with other chromosomes. Development of theseprotocols required optimization of a number of factors,including the amounts of probe and blocking DNA, the probeincubation time(s) prior to hybridization, and the time andconditions of hybridization. The following sections present asemiquantitative discussion of the considerations that led tothe protocols reported in this paper (mathematical details areavailable from the authors). Prehybridization of the targetcells with genomic DNA was also explored, but sufficientcontrast ratio can be obtained without it.

Hybridization without genomic DNA. Fig. la demon-strates the hybridization of pBS-4 DNA to a human meta-phase spread. All of the chromosomes are stained along mostof their lengths due to sequences in the probe that are sharedwith other chromosomes. Unstained regions, noted by ar-rows, show locations for which homologous sequences arenot present in pBS-4. These are mostly in the centromericregions and the long arm of the Y chromosome, where blocksof repetitive DNA specific to those sites are known to exist.The visible contrast on chromosome 4 is the result of theinteraction of several factors. (i) All of the DNA in chromo-some 4 is potential target for sequences in the probe, whereasonly those sequences on the other chromosomes that areshared with chromosome 4 can bind probe. (ii) The hybrid-ization time and probe concentration were high enough toallow significant binding of the specific sequences. (iii) Theratio of probe to target sequences is higher for the specificsequences than for the shared sequences. [Ten nanograms ofchromosome 4 DNA was hybridized to -200 ng of humanDNA target (4 x 104 cells), 13 ng of which is chromosome 4.Thus, the ratio of probe to target for the specific sequenceswas -1, whereas for the shared sequences it was =0.05.1The contrast can be increased by allowing the probe DNA

to partially rehybridize prior to adding it to the slide. Thispreferentially depletes the high-copy (predominantly theshared) sequences in the probe (20). A significant increase instaining specificity resulting from probe rehybridization wasobserved experimentally for chromosome 4 using a hybrid-ization mixture with 1 ng of probe per gl and a 24-hrincubation at 370C (not shown). Likewise, hybridization aftera 24-hr incubation of 4 ng of chromosome 21 probe per glresulted in a substantial contrast ratio. This indicates that atthese concentrations the chromosome-specific sequencesremain substantially single stranded for times on the order ofdays in the hybridization mixture. It also demonstrates thatother mechanisms that might inactivate the probe are notsignificant during the incubation.

Protocol I hybridization. The addition of unlabeled ge-nomic DNA to the hybridization mixture increases theconcentration of the shared sequences by a larger factor thanit increases the concentration of chromosome-specific se-quences. This enhances the preferential depletion of thelabeled copies of the shared sequences and increases thecontrast ratio. To see this, first consider one of the se-quences, repeat or unique, that is specific for the ith chro-mosome in a hybridization mixture containing a mass mp ofprobe DNA from the ith chromosome library and mb ofunlabeled genomic DNA. The number of labeled copies ofthesequence is proportional to mp. However, the number ofunlabeled copies is proportional to himb, where fi is thefraction of genomic DNA contained on the ith chromosome.Thus, Q, the ratio of unlabeled to labeled copies of each ofthesequences specific for the target chromosome, is fimb/mp.For human chromosomes, 0.016 < fi < 0.08 (21). For thework reported in this paper,f4= 0.066 andf2l = 0.016. Nowconsider a shared sequence that is distributed more-or-lessuniformly over the genome. The number of labeled copies isproportional to mp, whereas the number of unlabeled copiesis proportional to mb. Thus, the ratio of unlabeled to labeledcopies is mb/mp = Q/fi >> Q. This is true for all uniformlydistributed sequences, regardless of copy number. Thusadding genomic DNA increases the concentration of eachspecific sequence by the factor 1 + Q, whereas each uni-formly distributed sequence is increased by the larger factor1 + Qft.Although use of some genomic DNA is necessary to

generate a high contrast ratio, use of too much will decreasethe intensity of the specific staining to unacceptable levels.This is due to reassociation of the labeled specific sequencesin the probe and competition of the labeled and unlabeledcopies for hybridization sites. It can be shown that roughlyhalf of the beneficial effect of genomic DNA on relativerenaturation rates is achieved when Q = 1, and, by Q = 5,there is essentially no more benefit to be gained. Thus, ourprotocol I hybridizations keep Q < 5.

Results also improve if the hybridization time is sufficientlylong to give the specific sequences the opportunity to findtheir binding sites. Since there are more binding sites avail-able for the high-copy (shared) sequences than for thespecific sequences, staining of the non-target chromosomesoccurs more rapidly. Thus contrast improves as hybridiza-tion time is extended. Although we have no direct evidenceconcerning the type of sequence predominantly responsiblefor the chromosome-specific signals, Fig. lh indicates that wedo have the sensitivity to detect unique sequence hybridiza-tions of 3-kb target sites. However, low-copy chromosome-specific repeats (22, 23) may contribute to the hybridizationas well. If the unique sequences are predominantly respon-sible for the specific signals, then the concentration of eachsequence in our chromosome 4 hybridizations is about 10-100 times lower than that used with the group of 120chromosome 4 unique sequences. Thus one might expect thathybridization times >16 hr would be beneficial.

Fig. 1 b and c illustrate that very high contrast can beachieved by following these guidelines. A contrast ratio of-20 on chromosome 4 was obtained by using 1 ng of probeper IL, Q = 2 of genomic DNA, a 1-hr probe incubation, anda 110-hr hybridization. Weaker, but still adequate, signalswith a contrast ratio of =5 are achieved with a 16-hrhybridization under these conditions. Very bright signals canbe achieved with a 16-hr hybridization using 13.5 ng of probeper 1d and Q = 5 (Fig. le). The contrast is reduced relativeto that achieved in Fig. lb, but it is more than adequate.Experiments with increasing amounts of carrier DNA indi-cate that results become worse as the total DNA concentra-tion approaches 1 Ag/IA, so our protocols stay below thislimit.

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Proc. Natl. Acad. Sci. USA 85 (1988)

Protocol lI hybridization. Hybridization with pBS-21 DNAusing protocol I presents a problem not encountered withchromosome 4. This DNA hybridizes to chromosome 21 andto the centromeric regions of the D- and G-group chromo-somes. These regions contain ribosomal (19) and alphasatellite (24) sequences and perhaps others. These are rela-tively low-copy sequences shared with only a few chromo-somes, so protocol I is not very effective at suppressing themrelative to the chromosome 21-specific sequences. In addi-tion, these sequences are clustered on the chromosomes, sothat even much reduced hybridization is clearly visible. Thisis especially distracting in analysis of interphase nuclei.Calculations indicate that addition of several aliquots offreshly denatured genomic DNA periodically during theincubation (protocol II) should increase the staining speci-ficity. Fig. if shows a protocol II hybridization, using twoaliquots of genomic DNA, to a metaphase spread from atrisomy 21 cell line. Intense hybridization to the three number21 chromosomes is clearly visible and hybridization to theother D- and G-group chromosomes has been reduced to anacceptable level. Fig. lg shows that hybridization to chro-mosomes other than chromosome 21 is sufficiently low thatthe three chromosome 21 domains are clearly visible ininterphase nuclei. In practice, the most convenient procedurefor suppressing the shared acrocentric hybridization might beinclusion of unlabeled DNA from one of the other D- orG-group chromosome libraries (or unlabeled cloned DNAfrom just these sequences, if available) as additional com-petitor. The use of libraries from non-target chromosomes ascompetitor for a probe may in general improve contrast. Thespecific sequences in the probe will have no competition (Q= 0) no matter how much competitor for the shared se-quences is added.

Aberrant Chromosome Detection. Detection of numericaland structural aberrations with composite probes shouldfacilitate many areas of cytogenetics. Staining with thecomposite probe for chromosome 4 permits rapid identifica-tion of translocations in metaphase spreads (Fig. ld). Thehybridization intensity and specificity are such that even verysmall portions of the involved chromosome can be detected.This technique should be especially useful for applicationswhere high-quality banding is difficult (e.g., in tumor cyto-genetics) or where speed of analysis is the predominantconcern (e.g., for detection of low-frequency-induced chro-mosomal aberrations). Fig. le demonstrates the detection ofa homogeneously occurring translocation involving chromo-some 4 in interphase nuclei from a leukemia cell line (15)carrying the translocation t(4;11). Approximately half of thenuclei of this cell line showed three brightly fluorescentdomains, presumably produced by the two portions of theinvolved chromosome 4 and the intact normal chromosome.The domains in the other nuclei may have been obscured bythe nuclear orientation in these two-dimensional views, bynuclear distortion that occurred during slide preparation, orbecause the domains were too close to each other to bedistinguished. Hybridization using procedures that preservethree-dimensional morphology may resolve these issues andalso permit general studies of chromosomal domains ininterphase nuclei (ref. 4; unpublished).

Prenatal screening for disease-linked chromosome aberra-tions (e.g., trisomy 21) would be enhanced by techniques thatallow more rapid detection of such aberrations. Fig. 1 f andg show that competitive hybridization with pBS-21 DNAallows detection of trisomy 21 in metaphase spreads and ininterphase nuclei. Interphase aneuploidy analysis is particu-larly exciting since it will yield rapid results (cell culture is notnecessary). Whether the hybridization patterns are suffi-ciently reliable for routine prenatal diagnosis remains to bedetermined. Complications may result from clustering, over-

lap, and/or distortion of the fluorescent domains. However,these preliminary results are encouraging.Chromosome-specific libraries for all of the human chro-

mosomes are now available, so that production of a kit ofreagents capable of staining each of the human chromosomesshould be straightforward. The procedure also should workfor other species whose genomes have the same generalfeatures as the human. Staining of subchromosomal segmentsshould be possible using libraries containing those se-quences. Large insert libraries, cosmids and eventually yeastartificial chromosomes, are being produced for the humanchromosomes. These will facilitate selection of probes forchromosomal segments since single clones from these librar-ies can be hybridized to human chromosomes and nuclei withefficiencies approaching 100% (ref. 13; J. Landegent, unpub-lished data).We are grateful to Dr. C. Gilliam for providing the 120 chromo-

some 4-specific unique sequences, Dr. R. Schmickel for the ribo-somal clone pA, and Dr. J. Kersey for the RS(4;11) cell line. Wethank C. Lozes for preparing many of the cells and chromosomesused in this work and Drs. M. Andreeff and J. Haimi for the nucleifrom the RS(4;11) cells. This work was performed under the auspicesof the U.S. Department of Energy by the Lawrence LivermoreNational Laboratory under Contract W-7405-ENG-48 with supportfrom U.S. Public Health Service Grants HD 17655 and CA 45919.

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