High - resolution physical mapping by combined Alu - hybridization / PCR screening : Construction of a yeast artificial chromosome map covering 31 centimorgans in 3 p 21 - pl 4 ( Alu - PCR / chromosome 3 )

We describe an integrated approach to largescale physical mapping using an Alu-PCR hybridization screening strategy in conjunction with direct PCR-based screening to construct a continuous yeast artificial chromosome map covering >20 mb in human chromosome 3, bands pl4-p21, composed of 205 loci, connected by 480 yeast artificial chromosomes, with average interlocus distance of 100 kb. We observe an inverse distribution ofAlu-PCR and (CA)n markers. These results suggest that the two screening methods may be complementary and demonstrate the utility of AluPCR hybridization screening in the closure of high-resolution human physical maps. The predominant method for physical mapping of human chromosomes is based on the use of loci termed sequence tagged sites (STSs) to identify specific large-insert clones in libraries prepared from high-capacity DNA vectors such as yeast artificial chromosomes (YACs) through PCR-based screening of DNAs prepared from hierarchically pooled DNA samples from the library (1-3). Identification of overlapping sets of YACs by unique STSs allows the STSs to be ordered along chromosomes. STSs that detect polymorphic loci can be ordered on a genetic map, providing a basis for ordering contiguous groups of overlapping YAC clones (contigs). This approach has been used to assemble continuous overlapping YAC sets (YAC contigs) spanning most of chromosome arms 21q (3) and 22q (4) and the euchromatic portion of the Y chromosome (2). Higher density genetic and radiation hybrid maps promise to simplify the assembly of physical maps by providing a more extensive scaffolding for ordering YAC groups. Several non-STS-based methods have been developed for large-scale mapping, in an attempt to reduce the cost and effort involved. Principal among these are YAC fingerprinting by hybridization of medium copy repeat elements to digested YAC DNA (5) and hybridization of YAC Alu-PCR products to Alu-PCR products from pools of YACs (6). The high incidence of chimeric YACs in available libraries prevents sole use of either method in map construction. Rather, data produced by these methods is mainly useful in the context of more certain data produced by STS content mapping. We describe here the application of an Alu-PCR hybridization approach to physical mapping, in which individual AluPCR products are used as loci. This approach, combined with STS content mapping, was used to produce a continuous YAC map in 3p21-pl4 that links 205 loci and spans >20 mb with an average interlocus distance of -100 kb. The application of bothAlu-PCR product hybridization and STS content mapping to the same genomic region allowed comparison of the two methods. MATERIALS AND METHODS Cell 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 irradiationreduced human-hamster hybrids carrying portions of human chromosome 3 as their sole human content. HA54 contains 3pl4 and 3p25 (H.A. and D.E.H., unpublished results). DM1 and DG(2)3 (provided by S. Naylor, University of Texas, San Antonio) carry several discontinuous segments of 3p. T317, a human-hamster hybrid provided by W. E. C. Bradley (Hopital Notre-Dame, Montreal), contains 3pter-p21 as its sole human chromosome 3 content (7). YAC Library Screening. Centre d'Etude Polymorphisme Humain (CEPH) plates 1-550 (8) were screened by PCR analysis of hierarchically pooled YACs or by hybridization of individual Alu-PCR probes to Alu-PCR products of the YAC pools (9). CEPH plates 709-964, containing "megaYACs" (average insert -1 mb) (6, 10) were screened mainly by hybridization ofAlu-PCR probes toAlu-PCR products ofYAC pools. YAC pool DNAs were provided by T. Hudson and S. Foote (Whitehead-Massachusetts Institute of Technology Center for Genome Research). Alu-PCR. Alu-PCR products (11) were amplified from cosmid and YAC DNA templates (annealing temperature, 58°C) using four separateAlu-PCR reactions with primersAle 1,Alu S/J, Ale 3, andAlu END (9, 12). The latter three primers were also used to selectively amplify humanAlu-PCR products from human-rodent somatic cell hybrid DNAs (annealing temperature, 64°C).Alu-vectorette PCR products (12) were produced with Alu S/AluJ. L1-PCR was performed as described by Ledbetter et al. (13). Verification of YAC Probe Content. For STS verification miniprep DNA (14) was prepared from individual YACs for PCR analysis. Alu-PCR probes were verified by hybridization to dot-blotted Alu PCR products amplified from miniprep DNA of individual YACs. YAC End Isolation and STS Development. STSs were developed using DNA sequences from the ends of YAC or cosmid inserts amplified by vectorette PCR (15). Ends mapping to 3pl4 (see below) were cycle sequenced (Promega femtomole kit) with vector primers 1207 or 1208 (15). STSs were also made from exons andAlu or LI PCR products, which were cloned before sequencing. All sequences were screened against GenBank using BLAST (16) to eliminate repetitive sequences before designing oligonucleotide primers. RESULTS AND DISCUSSION Production of3p14 Probes. To isolate YAC clones spanning 3pl4 we collected entry probes from a variety of sources (Table Abbreviations: YAC, yeast artificial chromosome; STS, sequence tagged site; contig, contiguous group of overlapping clones; CEPH, Centre d'Etude Polymorphisme Humain; cM, centimorgan. *Present address: 3rd Department of Internal Medicine, University of Tokyo, Hongo, Tokyo 113, Japan. 4474 The publication costs of this article were defrayed in part by page charge payment. 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 93 (1996) 4475 1). Starting from many sites should facilitate early coalescence of large contigs and reduce the downstream effort required for extension of contigs and gap closure. Subsequent contig extension was accomplished by developing further probes (STSs or Alu-PCR products) from YACs at contig ends. Altogether 205 probes were used in this study. They fall into two heterogeneous groups: (i) 92 STSs used for PCR-based screening and (ii) 113 Alu-PCR or L1-PCR products used for hybridization-based screening. STS Probes. Fifty-eight of the 92 STSs are new. They were developed from genes, putative exons, cosmid ends, and YAC ends, as well as Alu, LI (13), and Alu-vectorette (12) PCR products. STSs were generated from three genes: PTPy (18), WNT5A (19), and MITF (20). Cosmids used for STS production came from libraries made from somatic-cell hybrids DM-1 and 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 were isolated in the course of contig extension from YACs at contig boundaries (see below) using vectorette PCR (15). Thirty STSs were polymorphic (CA)n repeats used in the CEPH/Genethon genetic map (22) or by Weber's group (23). They tether our physical map to the CEPH-Genethon genetic map. Alu-PCR Probes. One hundred and sixAlu-PCR hybridization probes were derived from somatic-cell hybrids [DG(2)3, DM-1, and HA54], cosmids [DM-1, HA54, and chromosome 3 (21) cosmid libraries], or 3pl4 YACs. Templates were amplified in three or four separate PCRs, each with one Alu primer or primer set: Alu S/J,Ale 1,Ale 3, orAlu END (9, 12). The use of several Alu primers increased the diversity of Alu-PCR products. AllAlu-PCR probes were treated as loci in the sense that single Alu-PCR products were used for hybridization screening of the YAC library. By avoiding the use of complex mixtures of Alu-PCR products (e.g., from a whole YAC) identification of chimeric YAC insert was simplified: the set of YACs detected by a single Alu-PCR product generally either fit in the contig or not. Library Screening. All probes, both STSs and Alu-PCR products, 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 of 92 STSs failed to detect at least one YAC address: D3S1300 and B2-A89. Alu-PCR hybridization screening of the CEPH I library yielded an average of 7.2 YACs per locus. Sixty-four percent of Alu hybridization screens produced one or more readable YAC addresses. In the contigs that emerged, STS and Alu-PCR loci are interspersed at high density (Fig. 1), demTable 1. Origin of loci Screening method Source STS Hybridization Total Cosmids 17 7 24 (CA), markers 30 0 30 Trinucleotide STSs 1 0 1 Anonymous 2 0 2 87 Genes, ESTs 4 0 4 Entry probes L1-PCR products 1 0 1 Entry bes Alu-PCR products From hybrids 6 19 25 From YACs 2 87 891 YAC insert ends 32 0 32 Walking probes Totals 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). Two other cosmid derived STSs contained short tandem repeats and are grouped with the 32 (CA), repeats. EST, expressed sequence tags. onstrating sufficient correspondence in YAC address detection 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 the margins of contigs into STSs and screening YAC libraries or by isolating individual Alu-PCR products from YACs at the edges of contigs and using them to screen YAC libraries by hybridization. In general, hybridization was a faster method for contig extension. Overall 89 YAC-derived Alu-PCR products and 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 chimeric YACs. Accordingly, YAC end fragments were localized to 3pl4 before STS conversion using somatic-cell hybrids HA54 and T317. HA54 carries 3pl4 and 3p25 as its sole human content. T317, which contains 3pter-p21 as its sole human chromosome 3 content (7), was used to assign HA54-positive probes to either the 3p25 (if positive in T317) or 3pl4 (if negative in T317) segment of HA54. We chose the 3pl4 segment of HA54 as our target region for YAC coverage. To eliminate non-3p14 Alu-PCR products from consideration as probes, Alu-PCR products from YACs with overlapping inserts were analyzed in adjacent wells of agarose gels. Alu-PCR products identical in size and amplified from two or more overlapping YACs were considered likely to be the same molecule and therefore likely (given the random origin of the non-3p14 content of chimeric YACs) to arise from 3pl4 YAC insert. YAC Contig Assembly. After screening the CEPH I library 10 contigs ranging in length from 1 to 5 mb were obtained. The contigs were ordered and oriented based on the location of (CA), loci from the CEPH-Genethon genetic map (22). The final nine gaps were closed by screening the CEPH megaYAC library, which contains human inserts over twice as long as those in the CEPH I library (6). Selected Alu-PCR products were used to screen the CEPH megaYAC library by hybridization to port the CEPH I library contigs to the CEPH megaYAC library. To fill the gaps between megaYAC contigs, contig extension was done by either YAC end isolation or Alu-PCR hybridization. The two last gaps were closed by D3S2400 and by data from Whitehead Institute/Massachusetts Institute of Technology (MIT) anonymous DNA markers D3S2478 and D3S2483. The final contig spans the p14 segment of HA54, which by fluorescence in situ hybridization is estimated to be >20 mb (M. Haas and D. Ward, personal communication). The outermost genetic markers, D3S1581 (telomeric) and D3S1598 (centromeric), subtend 31 centimorgans (cM) in the CEPH genetic map (22). The contig consists of 480 YAC clones: 291 from the CEPH1 and 189 from the megaYAC library. One hundred fifty four of 205 DNA markers are ordered unambiguously. The average interlocus distance is 100 kb. Integration with Other Mapping Data. MegaYAC screening data for most of the (CA)n repeat loci was obtained from CEPH/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 considerable use in assembling our contig. Full information for the loci that make up this map is available at the University of Texas at San Antonio Chromosome 3 World Wide Web site (http://mars.uthscsa.edu/DB/). Distribution ofAlu Markers vs. (CA)n Repeat Markers. The location of somatic-cell hybrid-derived Alu-PCR entry probes and (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, with Alu clustering more pronounced. The two classes of probes appear inversely distributed, particularly when contigs derived from data produced by each method alone in the CEPH I library are compared (Fig. 2, columns F and H). Column F Genetics: Aburatani et al. 4476 Genetics: Aburatani et al. I I I I I I I Proc. Natl. Acad. Sci. USA 93 (1996)