Proc. Natl. Acad. Sci. USAVol. 93, pp. 4474-4479, April 1996Genetics

High-resolution physical mapping by combined Alu-hybridization/PCR screening: Construction of a yeast artificial chromosomemap covering 31 centimorgans in 3p21-pl4

(Alu-PCR/chromosome 3)

HIROYUKI ABURATANI*, VINCENT P. STANTON, JR., AND DAVID E. HOUSMANCenter for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by David E. Housman, November 29, 1995

ABSTRACT We describe an integrated approach to large-scale physical mapping using an Alu-PCR hybridizationscreening strategy in conjunction with direct PCR-basedscreening to construct a continuous yeast artificial chromo-some map covering >20 mb in human chromosome 3, bandspl4-p21, composed of 205 loci, connected by 480 yeast arti-ficial chromosomes, with average interlocus distance of 100kb. We observe an inverse distribution ofAlu-PCR and (CA)nmarkers. These results suggest that the two screening methodsmay be complementary and demonstrate the utility of Alu-PCR hybridization screening in the closure of high-resolutionhuman physical maps.

The predominant method for physical mapping of humanchromosomes is based on the use of loci termed sequencetagged sites (STSs) to identify specific large-insert clones inlibraries prepared from high-capacity DNA vectors such asyeast artificial chromosomes (YACs) through PCR-basedscreening of DNAs prepared from hierarchically pooled DNAsamples from the library (1-3). Identification of overlappingsets of YACs by unique STSs allows the STSs to be orderedalong chromosomes. STSs that detect polymorphic loci can beordered on a genetic map, providing a basis for orderingcontiguous groups of overlapping YAC clones (contigs). Thisapproach has been used to assemble continuous overlappingYAC sets (YAC contigs) spanning most of chromosome arms21q (3) and 22q (4) and the euchromatic portion of the Ychromosome (2). Higher density genetic and radiation hybridmaps promise to simplify the assembly of physical maps byproviding a more extensive scaffolding for ordering YACgroups.

Several non-STS-based methods have been developed forlarge-scale mapping, in an attempt to reduce the cost andeffort involved. Principal among these are YAC fingerprintingby hybridization of medium copy repeat elements to digestedYAC DNA (5) and hybridization of YAC Alu-PCR productsto Alu-PCR products from pools of YACs (6). The highincidence of chimeric YACs in available libraries prevents soleuse of either method in map construction. Rather, dataproduced by these methods is mainly useful in the context ofmore certain data produced by STS content mapping.We describe here the application of an Alu-PCR hybridiza-

tion approach to physical mapping, in which individual Alu-PCR products are used as loci. This approach, combined withSTS content mapping, was used to produce a continuous YACmap in 3p21-pl4 that links 205 loci and spans >20 mb with anaverage interlocus distance of -100 kb. The application ofbothAlu-PCR product hybridization and STS content mappingto the same genomic region allowed comparison of the twomethods.

MATERIALS AND METHODSCell Lines. A9(Neo 3/t) is a human-mouse hybrid cell line

monochromosomal for human 3 (provided by M. Oshimura,Tottori University). HA54, DM-1, and DG(2)3 are irradiation-reduced human-hamster hybrids carrying portions of humanchromosome 3 as their sole human content. HA54 contains3pl4 and 3p25 (H.A. and D.E.H., unpublished results). DM1and DG(2)3 (provided by S. Naylor, University of Texas, SanAntonio) carry several discontinuous segments of 3p. T317, ahuman-hamster hybrid provided by W. E. C. Bradley (HopitalNotre-Dame, Montreal), contains 3pter-p21 as its sole humanchromosome 3 content (7).YAC Library Screening. Centre d'Etude Polymorphisme

Humain (CEPH) plates 1-550 (8) were screened by PCRanalysis of hierarchically pooled YACs or by hybridization ofindividual Alu-PCR probes to Alu-PCR products of the YACpools (9). CEPH plates 709-964, containing "megaYACs"(average insert -1 mb) (6, 10) were screened mainly byhybridization ofAlu-PCR probes toAlu-PCR products ofYACpools. YAC pool DNAs were provided by T. Hudson and S.Foote (Whitehead-Massachusetts Institute of TechnologyCenter for Genome Research).Alu-PCR. Alu-PCR products (11) were amplified from cos-

mid and YAC DNA templates (annealing temperature, 58°C)using four separateAlu-PCR reactions with primersAle 1,AluS/J, Ale 3, andAlu END (9, 12). The latter three primers werealso used to selectively amplify humanAlu-PCR products fromhuman-rodent somatic cell hybrid DNAs (annealing temper-ature, 64°C).Alu-vectorette PCR products (12) were producedwith Alu S/AluJ. L1-PCR was performed as described byLedbetter et al. (13).

Verification of YAC Probe Content. For STS verificationminiprep DNA (14) was prepared from individual YACs forPCR analysis. Alu-PCR probes were verified by hybridizationto dot-blotted Alu PCR products amplified from miniprepDNA of individual YACs.YAC End Isolation and STS Development. STSs were de-

veloped using DNA sequences from the ends of YAC orcosmid inserts amplified by vectorette PCR (15). Ends map-ping to 3pl4 (see below) were cycle sequenced (Promegafemtomole kit) with vector primers 1207 or 1208 (15). STSswere also made from exons andAlu or LI PCR products, whichwere cloned before sequencing. All sequences were screenedagainst GenBank using BLAST (16) to eliminate repetitivesequences before designing oligonucleotide primers.

RESULTS AND DISCUSSIONProduction of3p14 Probes. To isolate YAC clones spanning

3pl4 we collected entry probes from a variety of sources (Table

Abbreviations: YAC, yeast artificial chromosome; STS, sequencetagged site; contig, contiguous group of overlapping clones; CEPH,Centre d'Etude Polymorphisme Humain; cM, centimorgan.*Present address: 3rd Department of Internal Medicine, University ofTokyo, Hongo, Tokyo 113, Japan.

4474

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Proc. Natl. Acad. Sci. USA 93 (1996) 4475

1). Starting from many sites should facilitate early coalescenceof large contigs and reduce the downstream effort required forextension of contigs and gap closure. Subsequent contig ex-tension was accomplished by developing further probes (STSsor Alu-PCR products) from YACs at contig ends. Altogether205 probes were used in this study. They fall into two heter-ogeneous groups: (i) 92 STSs used for PCR-based screeningand (ii) 113 Alu-PCR or L1-PCR products used for hybridiza-tion-based screening.STS Probes. Fifty-eight of the 92 STSs are new. They were

developed from genes, putative exons, cosmid ends, and YACends, as well as Alu, LI (13), and Alu-vectorette (12) PCRproducts. STSs were generated from three genes: PTPy (18),WNT5A (19), and MITF (20). Cosmids used for STS produc-tion came from libraries made from somatic-cell hybrids DM-1and HA54 and from a chromosome 3-specific library (21).Putative exons were spliced from six cosmids: cCI825, cCI912,cCI948, cCI1416, cCI1430, and D3S1149 (17). YAC ends wereisolated in the course of contig extension from YACs at contigboundaries (see below) using vectorette PCR (15).

Thirty STSs were polymorphic (CA)n repeats used in theCEPH/Genethon genetic map (22) or by Weber's group (23).They tether our physical map to the CEPH-Genethon geneticmap.Alu-PCR Probes. One hundred and sixAlu-PCR hybridiza-

tion probes were derived from somatic-cell hybrids [DG(2)3,DM-1, and HA54], cosmids [DM-1, HA54, and chromosome3 (21) cosmid libraries], or 3pl4 YACs. Templates wereamplified in three or four separate PCRs, each with one Aluprimer or primer set: Alu S/J,Ale 1,Ale 3, orAlu END (9, 12).The use of several Alu primers increased the diversity ofAlu-PCR products. AllAlu-PCR probes were treated as loci inthe sense that single Alu-PCR products were used for hybrid-ization screening of the YAC library. By avoiding the use ofcomplex mixtures of Alu-PCR products (e.g., from a wholeYAC) identification of chimeric YAC insert was simplified: theset of YACs detected by a single Alu-PCR product generallyeither fit in the contig or not.

Library Screening. All probes, both STSs and Alu-PCRproducts, were screened against the CEPH I YAC library,which contains approximately seven genomes of human insert(8). An average of 5.7 YACs was detected per STS. Only 2 of92 STSs failed to detect at least one YAC address: D3S1300and B2-A89. Alu-PCR hybridization screening of the CEPH Ilibrary yielded an average of 7.2 YACs per locus. Sixty-fourpercent of Alu hybridization screens produced one or morereadable YAC addresses. In the contigs that emerged, STS andAlu-PCR loci are interspersed at high density (Fig. 1), dem-

Table 1. Origin of loci

Screening method

Source STS Hybridization Total

Cosmids 17 7 24(CA), markers 30 0 30Trinucleotide STSs 1 0 1Anonymous 2 0 2 87Genes, ESTs 4 0 4 Entry probesL1-PCR products 1 0 1 Entry besAlu-PCR productsFrom hybrids 6 19 25From YACs 2 87 891

YAC insert ends 32 0 32 Walking probesTotals 92 113 205

Probes used to make the 3p14 physical map sorted by source DNA,YAC screening method, and use (entry probes vs. walking probes).Four of the six hybrid-derived STSs are Alu-vectorette PCR products(12). The 17 cosmid-derived STSs include six putative exons (17). Twoother cosmid derived STSs contained short tandem repeats and aregrouped with the 32 (CA), repeats. EST, expressed sequence tags.

onstrating sufficient correspondence in YAC address detec-tion between the two methods to allow contig assembly.YAC Contig Extension. YAC contig extension in the CEPH

I library was accomplished by converting YAC ends at themargins of contigs into STSs and screening YAC libraries orby isolating individual Alu-PCR products from YACs at theedges of contigs and using them to screen YAC libraries byhybridization. In general, hybridization was a faster method forcontig extension. Overall 89 YAC-derived Alu-PCR productsand 32 YAC ends were used successfully in contig extension.Whether PCR or hybridization screening was used, it was

desirable to avoid probes from non-3p14 segments of chimericYACs. Accordingly, YAC end fragments were localized to3pl4 before STS conversion using somatic-cell hybrids HA54and T317. HA54 carries 3pl4 and 3p25 as its sole humancontent. T317, which contains 3pter-p21 as its sole humanchromosome 3 content (7), was used to assign HA54-positiveprobes to either the 3p25 (if positive in T317) or 3pl4 (ifnegative in T317) segment of HA54. We chose the 3pl4segment of HA54 as our target region for YAC coverage.To eliminate non-3p14 Alu-PCR products from consider-

ation as probes, Alu-PCR products from YACs with overlap-ping inserts were analyzed in adjacent wells of agarose gels.Alu-PCR products identical in size and amplified from two ormore overlapping YACs were considered likely to be the samemolecule and therefore likely (given the random origin of thenon-3p14 content of chimeric YACs) to arise from 3pl4 YACinsert.YAC Contig Assembly. After screening the CEPH I library

10 contigs ranging in length from 1 to 5 mb were obtained. Thecontigs were ordered and oriented based on the location of(CA), loci from the CEPH-Genethon genetic map (22). Thefinal nine gaps were closed by screening the CEPH megaYAClibrary, which contains human inserts over twice as long asthose in the CEPH I library (6). Selected Alu-PCR productswere used to screen the CEPH megaYAC library by hybrid-ization to port the CEPH I library contigs to the CEPHmegaYAC library. To fill the gaps between megaYAC contigs,contig extension was done by either YAC end isolation orAlu-PCR hybridization. The two last gaps were closed byD3S2400 and by data from Whitehead Institute/Massachu-setts Institute of Technology (MIT) anonymous DNA markersD3S2478 and D3S2483. The final contig spans the p14 segmentof HA54, which by fluorescence in situ hybridization is esti-mated to be >20 mb (M. Haas and D. Ward, personalcommunication). The outermost genetic markers, D3S1581(telomeric) and D3S1598 (centromeric), subtend 31 centimor-gans (cM) in the CEPH genetic map (22). The contig consistsof 480 YAC clones: 291 from the CEPH1 and 189 from themegaYAC library. One hundred fifty four of 205 DNA mark-ers are ordered unambiguously. The average interlocus dis-tance is 100 kb.

Integration with Other Mapping Data. MegaYAC screen-ing data for most of the (CA)n repeat loci was obtained fromCEPH/Genethon (http://www.cephb.fr/ceph-genethonmap.html) and Whitehead/MIT Genome Center (http://www.genome.wi.mit.edu/) data bases. These data were of consid-erable use in assembling our contig. Full information for theloci that make up this map is available at the University ofTexas at San Antonio Chromosome 3 World Wide Web site(http://mars.uthscsa.edu/DB/).

Distribution ofAlu Markers vs. (CA)n Repeat Markers. Thelocation of somatic-cell hybrid-derived Alu-PCR entry probesand (CA)n repeat probes in the 3pl4 contig is shown in Fig. 2.Both the Alu-derived entry probes (circles, column E) and the(CA)n repeat STSs (column G) are unevenly distributed, withAlu clustering more pronounced. The two classes of probesappear inversely distributed, particularly when contigs derivedfrom data produced by each method alone in the CEPH Ilibrary are compared (Fig. 2, columns F and H). Column F

Genetics: Aburatani et al.

4476 Genetics: Aburatani et al.

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FIG. 1. YAC map of 3p21-pl4. Two hundred and five markers that detect 480 YACs are ordered in 154 bins. All 92 STS markers are markedwith a solid rectangle at top. YACs are divided in two groups: a CEPH I group above and a CEPH megaYAC group below. Markers used to screenthe CEPH I library are shown above the CEPH I YACs. Markers used to screen the megaYAC library are shown above the megaYACs. A subsetof the markers used to screen the megaYAC library (those aligned toward the CEPH I YACs) were also used to screen the CEPH I YACs. Markersused only to screen the megaYAC library are aligned toward the megaYACs. Stippled lines indicate probable internal deletions in YACs.Parenthetical YAC coordinates indicate ambiguity or contamination. Proven nonchimeric insert ends are marked by black circles.

Proc. Natl. Acad. Sci. USA 93 (1996)

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A, the 3p21.1-p14 segment of hybrid HA54 with two internal deletionsshown lightly shaded. Column B, genetic linkage map by Genethon (22).Column C, genetic map by others (23). The vertical scale is geneticdistance. Columns D-K, results with CEPH I library. Column D, non-Alu,non-(CA), entry probes (empty circles). Column E, Alu-related entryprobes, shown by filled circles (e). Column F, YAC contigs that could beassembled solely from Alu-related entry probes (black bars). Column G,(CA), markers (filled circles). Column H, contigs that could be assembledsolely from (CA), markers (gray bars). Column J, contigs that could beassembled from both (CA),, markers and Alu-related entry probes.Column K, CEPH I YAC contigs after contig extension. Column L,megaYAC contigs that could be assembled solely from (CA)n markers.Column M, megaYAC contig after integration with CEPH I data andfurther contig extension.

shows the 15 contigs that could be assembled with the datafromAlu entry markers alone. Column H shows the 15 contigs

that could be assembled solely with YACs detected by (CA)nmarkers. Column J shows the 16 contigs that can be assembledfrom a combined probe collection. Only 2 of the 15 (CA)n-derived contigs lie entirely within Alu-derived contigs. Theother 13 (CA),-derived contigs extend (10 contigs) or lieentirely outside (3 contigs) the Alu-derived contigs.Both Alu elements and (CA)n repeats are unevenly distrib-

uted along chromosomes. Alu elements are reported to beconcentrated in R bands (24), whereas (CA)n repeats aresparse in subtelomeric regions (25). The distribution of Alusand (CA)n repeats at the submegabase scale has not beeninvestigated in detail. Fig. 2 shows the distribution of randommembers of these two classes of elements over a -31-cMsegment. The inverse distribution of the two classes of probessuggests that closure of YAC contigs derived primarily from(CA)n markers may be facilitated by use ofAlu-PCR product-derived markers. The success of Alu-PCR hybridization in3pl4, a Giemsa dark band comparatively underpopulated withAlu repeats (24) suggests the approach is sufficiently durableto work in virtually any genomic region (9). Of equal interest,although an inverse distribution of Alu repeats and (CA)nrepeats is observed at the submegabase level, from the per-spective of a genetic map Alu and (CA)n markers are exten-sively interspersed throughout 3p14. The conversion of Alu-based polymorphisms into genetic markers would thus bepredicted to lead to an integrated genetic map in whichAlu-based markers would alternate with (CA)n markers at adensity well below 1 cM.

Note Added in Proof. Two other high resolution physical maps of the3p14 region are described in refs. 26 and 27.

We acknowledge the contributions of the CEPH/Genethon groupsled by J. Weissenbach, I. Chumakov, and D. Cohen, whose YAClibraries and genetic map were essential for our work. We thank H.Kanki, K. Ohira, J. Takeyama, C. Guydan, J. Bernadini, S. Ontiveros,J. M. Andresen, J. Warren, B. Freeman, M. Bulyk, and A. Bany fortechnical assistance. We also thank K. Yamakawa and Y. Nakamura(cosmids), D. J. Munroe (Alu-bubble PCR), and M. Haas and D. Ward(fluorescence in situ hybridization). This work was supported byNational Institutes of Health Grants HG 00299 and CA 17575 toD.E.H.

1. Green, E. D. & Olson, M. V. (1990) Proc. Natl. Acad. Sci. USA87, 1213-1217.

2. Foote, S., Vollrath, D., Hilton, A. & Page, D. C. (1992) Science258, 60-66.

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