Proc. Nati. Acad. Sci. USA Vol. 86, pp. 5030-5034, July 1989 Genetics Physical mapping of complex genomes by cosmid multiplex analysis (genomic cloning/chromosome li/genomic data base/human genome/DNA fingerprinting) G. A. EVANS* AND K. A. LEWIS Molecular Genetics Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037 Communicated by Renato Dulbecco, March 27, 1989 (receivedfor review January 20, 1989) ABSTRACT A rapid and powerful approach for linking In this paper, we describe an alternate strategy for bottom- individual clones ofa cosmid library and the assembly ofa large up mapping that is applicable to the analysis of mammalian physical map is presented, which depends on the simultaneous chromosomes and allows for the simultaneous determination analysis of many cosmid clones for overlapping regions. This ofoverlaps between cosmid clones analyzed in pools. Rather method uses cosmid vectors that contain endogenous bacteri- than depending on fingerprinting procedures for detection of ophage T3 and T7 promoters to allow for the identification of overlapping clones, we constructed cosmid libraries by using overlapping clones through the synthesis of end-specific RNA vectors containing T3 or T7 bacteriophage promoters flank- probes. A genomic library is constructed and organized as an ing the cloned genomic DNA (10, 11) such that overlapping ordered matrix such that each done is assigned an identifying sequences could be detected by hybridization. To test the coordinate. DNA from mixtures ofcosmid clones is pooled such feasibility of this strategy, we analyzed 960 clones isolated that each pool contains only one common member with any from human chromosome 11 for overlapping regions by other pool, RNA probes are prepared from mixtures of cosmid preparing 68 mixed RNA probes, which were used for clones, and groups of clones overlapping with the constituents hybridization to replicas of this filter. This simple procedure ofthe mixtures are determined by hybridization. Pooled probes resulted in the ordering of cosmid clones spanning 11q13- are most simply prepared by grouping clones according to the llqter and the identification ofcontigs containing many ofthe rows and columns of the library matrix. The pairwise com- genes mapping to this chromosome. This method is poten- parison ofdata generated by the hybridization ofmixed probes tially applicable to cloning, ordering clones, and physical can be decoded by using simple algorithms that predict the mapping of other complex genomes as well. order and linkage of all clones in the collection and organize them into predicted contigs. To demonstrate the feasibility of multiplexed analysis of cosmids, a genomic library was pre- MATERIALS AND METHODS pared from a mouse-human somatic cell hybrid that contains Cell Lines. TG 5D1-1 is a Friend cell line derived from a portion of the long arm of human chromosome 11. Prepa- somatic cell hybrid 5D1 [which carries an intact human X ration, arrangement on a matrix, and analysis ofpooled cosmid chromosome and chromosome 11 (12)] and selected with clones from this collection resulted in the detection of 1099 6-thioguanine for the loss of the entire X chromosome and linked pairs of cosmids, which could be assembled into 315 most ofchromosome 11. TG 5D1-1 contains the distal portion contigs. Thus, with a minimal amount of effort, a substantial of chromosome 11 as the only human material in a mouse portion of this genomic region has been linked in multiple genomic background (13), representing about 0.9o of the overlapping contigs. This method may have practical applica- human genome. tions in the large-scale mapping and sequencing of mammalian Cosmid Vectors and Libraries. Genomic libraries were genomes. constructed in cosmid vector sCos-1 (11), which contains duplicated cos sites for high efficiency microcloning, T3 and The analysis of large genomes will require the application of T7 bacteriophage promoters flanking the unique BamHI both "top-down" and "bottom-up" mapping strategies. The cloning site, two Not I sites for the excision of genomic former strategy depends on the separation on pulsed-field inserts, a selectable gene (SV2-neo9 for mammalian gene DNA rare transfer, and a ColEl origin of replication (see Fig. 1). gels of large fragments generated by using restric- Detailed restriction maps of the cosmid insert in this vector tion endonucleases for physical linkage ofDNA markers and may be rapidly determined by an end-labeling mapping the construction oflong-range maps (1-4). The latter strategy procedure using T3- or T7-specific oligonucleotides (11, 14, depends on identifying overlapping sequences in a large 15). The genomic cosmid library used in this study consisted number ofrandomly selected bacteriophage or cosmid clones of 1.5 x 107 independent clones and was constructed by using by unique restriction enzyme "fingerprinting" (4, 5) and their genomic DNA digested to an average size of 100-120 kilo- assembly into overlapping sets of clones referred to as bases, dephosphorylated with calf intestinal phosphatase, contigs (6). A similar strategy is potentially feasible using and packaged with Gigapak Gold (Stratagene) in vitro pack- megabase-size fragments cloned as yeast artificial chromo- aging lysate (11). Only nonamplified libraries were used, and somes (3). In the past few years, bottom-up mapping strat- cosmid clones were archived in 96-well microtiter plates egies have been successfully applied to generate complete or stored at -700C in LB media with 15% (vol/vol) glycerol and partial genomic maps of Saccharomyces cerevisiae (4), Cae- kanamycin sulfate at 25 pug/ml. norhabditis elegans (5, 7), and Escherichia coli (8). Poustka Library Screening. Cosmid libraries were plated on 576- et al. (9) proposed a different strategy for the ordering of cm2 LB agar trays at a density of 10 clones per cm2, replica cosmid or phage clones organized at high density on a matrix filters were prepared, and filters were hybridized with human by using identifying sequences detected with short oligonu- placenta DNA labeled with [32P]dCTP to a specific activity of cleotide probes. 108 cpm/,ug. Under these hybridization conditions, no back- The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Molecular payment. This article must therefore be hereby marked "advertisement" Genetics Laboratory, The Salk Institute, P.O. Box 85800, San in accordance with 18 U.S.C. §1734 solely to indicate this fact. Diego, CA 92138. 5030 Downloaded by guest on October 1, 2021 Genetics: Evans and Lewis Proc. Natl. Acad. Sci. USA 86 (1989) 5031 ground hybridization was detected against cosmids carrying Notl mouse genomic DNA. Cosmids containing human genomic T3 Promoter DNA inserts were picked with toothpicks, rescreened by BamHl hybridization to 32P-labeled human DNA, and transferred to 96-well archive microtiter plates. Replica transfer ofclones in 96-well microtiter plates and transfer from archived plates to screening filters was carried out with an aluminum "hedge- hog" (5) or a simple laboratory robot (Beckman Biomek 1000). For multiplex analysis, archived cosmids were inoc- ColEl ori sCos-1 ulated on the surface of a nitrocellulose- or nylon-based filter in a matrix or grid pattern using a 36 x 36 matrix based on the size and spacing of the 96-well archive plates. The clones were allowed to grow on the surface of the filter at 370C for 12-15 hr, and bacterial DNA was fixed to the filter by using a standard colony lysis procedure (16). RNA Probe Synthesis and Hybridization Reactions. (Knr ) Cosmids were transferred from archives to fresh 96-well plates containing liquid LB media with kanamycin sulfate at FIG. 1. A vector for cosmid multiplex analysis. The vector 25 Ag/ml and incubated at 370C in a humidified atmosphere sCos-1 contains bacteriophage T3 and T7 promoters flanking a for 6-10 hr. Supernatants from individual wells were pooled, unique BamHI cloning site, Not I sites for expedited restriction and DNA was prepared by using a cosmid minilysate pro- mapping and excision ofthe insert DNA, duplicated cos sites for high cedure (14). RNA probes were synthesized as previously efficiency microcloning, a dominant selection for transfection into described using bacteriophage T3 or T7 polymerase (Strata- mammalian cells (SV2neo9), ampicillin (Amp') and kanamycin (KnD) gene), and [32P]UTP and polymerase reactions were termi- resistance genes, and ColEl origin of replication (11). nated by extraction with phenol and chloroform. The RNA probe was prehybridized with a blocking mixture (a mixture 0.5% (10) and provide strategies for the simultaneous analysis of sonicated human placenta DNA and cloned human repet- of pools of clones. itive sequences at a concentration of 1 mg/ml) as described Overlapping contiguous cosmid clones arranged on an (11) and then hybridized to a replica of the matrix filter for organized matrix may be detected by the synthesis of an 12-18 hr under previously described conditions (11). Filters end-specific RNA probe and hybridization of the probe to a were washed in 0.lx SSC (0.015 M NaCl/0.0015 M sodium replica of the matrix filter. In addition, the use of hybridiza- citrate, pH 7.6)/0.1% SDS at 650C and exposed to x-ray film tion to detect overlapping clones allows a multiplex strategy for 2-8 hr. Restriction enzyme analysis of isolated cosmids where the RNA probes are prepared from predetermined were carried out by using labeled oligonucleotides recogniz- pools of cosmid templates. The templates are pooled such ing the T3 or T7 promoter sequences as described (11, 15). that each two pools contain only one cosmid in common; Data Analysis. The grid coordinates of hybridizing cosmid thus, comparison of the results of hybridization of two clones with each pool ofprobes were entered into a computer different probes to a matrix filter will ensure that clones file and analyzed by using computer programs written by detected by both pools represent that hybridizing to the G.A.E.