PCR amplification and analysis of yeast artificial chromosomes

SHORT COMMUNICATION PCRAmplification

and Analysis of Yeast Artificial Chromosomes

JAMESS. SUTCLIFFE,*FUPINGZHANG,* C. THOMASCAsKEY,t DAVID L. NELSON,+AND STEPHEN T. WARREN*,’ *Howard Hughes Medical Institute and Departments of Biochemistry and Pediatrics, Division of Medical Genetics, Emory University School of Medicine, Atlanta, Georgia 30322; $lnstitute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030; and tlnstitute for Molecular Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030 Received

October22,

1991;revised

A strategy for the analysis of yeast artificial chromosome (YAC) clones that relies on polymerase chain reaction (PCR) amplification of small restriction fragments from isolated YACs following adapter ligation was developed. Using this method, termed YACadapt, we have amplified several YACs from a human Xq24-qter library and have used the PCR products for physical mapping by somatic cell hybrid deletion analysis and fluorescent in situ hybridization. One YAC, RS46, was mapped to band Xq27.3, near the fragile X mutation. The PCR product is an excellent renewable source of YAC DNA for analyses involving hybridization of YAC inserts to a variety of DNA/RNA sources. o 1992 Academic Press, Inc.

The construction of comprehensive libraries in yeast artificial chromosome (YAC) vectors extends the analysis of cloned DNA into megabase-sized regions of complex genomes (2). As such, YAC technology addresses a critical resolution gap between standard cloning techniques and genetic mapping. Localizing YAC clones to specific chromosomes or chromosomal regions has facilitated high-resolution mapping within defined cytogenetic landmarks and is essential for genome analysis or the positional cloning of disease genes. However, YACs are less amenable than phage or cosmid clones to convenient manipulation. Although a variety of different approaches have been developed to facilitate end rescue and isolation of discrete internal fragments to generate probes (4,5,10,13) none surveys the entire YAC clone, limiting the identification of potentially important information within an insert. We describe a strategy for the PCR amplification of small restriction fragments generated from isolated YACs that is shown to be useful for precise physical mapping of the YAC. To test this strategy, we examined clones that had been previously isolated from a YAC library constructed from the hybrid cell X3000-11.1 containing Xq24-qter as its only human component (5). YAC clones, prepared within agarose plugs as described (l), were resolved in several lanes each through 1% low-melting (Seaplaque, FMC) agarose [preparative pulsed-field gels run with 5-s ’ To whom

correspondence

should

be addressed. 1303

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pulses for 12 h followed by 10-s pulses for 12 h at 200 V, 14°C in 0.5~ TBE (45 m&4 Tris-borate, 1 n&f EDTA) using a Bio-Rad CHEF DRII apparatus]. Bands corresponding to the YACs were sterilely excised following ethidium bromide visualization and transferred to microfuge tubes. The agarose was melted at 65°C and diluted 1:3 with water, and the DNA was digested to completion with 50 units Sau3AI (BRL) in its recommended buffer for 1 h at 37°C. Sau3AI fragments were recovered by ethanol precipitation following extraction with warm phenol, phenol/chloroform (l/l, v/v), and chloroform. The recovered DNA was ligated to phosphorylated, double-stranded oligonucleotide adapters in a 5:l molar end excess that contained a Sau3AI complementary overhang on one end (Fig. l), similar to a technique previously described (3). [Adapters were prepared by mixing at room temperature equimolar amounts of the corresponding single-stranded oligonucleotides, previously phosphorylated with T4 polynucleotide kinase (BRL) under established conditions (S)]. The 54-bp adapter dimer, formed by adapter self-ligation, was removed using a Centriconmicroconcentrator (Amicon) that retains fragments greater than 100,000 Da, thus allowing sequences less than 150 bp to pass through after cycles (e.g., four) of concentration and dilution (0.1 to 2.0 ml with ddH,O), retaining most Suu3AI fragments. The sequence of the adapters immediately adjacent to the Sau3AI site is complementary to the universal hgtl0 forward primer (Fig 1). PCR amplification in buffer containing 10 mM Tris-HCl, pH 8.3,1.5 mh4 MgCl,, 50 mM KCl, 0.01% gelatin, 200 pJ4 dNTPs, 50 pmol primer, and 2.5 units AmpliTaq polymerase (PerkinElmer) in a loo-p1 reaction with 60 cycles of 94”C-1 min, 55’C-1 min, and 72”C-1 min resulted in the observation of a smear of product ranging from 150 to about 2000 bp when resolved on a 1.5% agarose gel and is consistent with the size expected for a Sau3AI digest of genomic DNA (Fig. 2). One hundred nanograms of PCR product was purified from its flanking linkers by SuuSAI digestion and Centricon-100 filtration, labeled with 32P by nick-translation (7), and analyzed by Southern blot hybridization (12) to a somatic cell hybrid panel containing human, Chinese hamster ovary, and hybrid DNAs containing varying amounts of the human X chromosome (Fig. 3a). 13,1303-1306 (19%) osss-7543/92 $5.00 Copyright 0 1992 hy Academic Press, Inc. All rights of reproduction in any form reserved. GENOMICS

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Digest gel-purified

YAC with Sau ZA to completton

t (;ATC-(ITA(; Ligate with excess YACdapt

Denature I h gt IO left am, primer PCR amplify

5’ A(;CAA(;TTCA(;CCT(;(;TTAA(;

FIG. 1. Overall strategy for the amplification sive end is used to flank Sau3AI fragments from adapting molecules.

of yeast a purified

artificial chromosome YAC. XgtlO forward

FIG. 2. Agarose gel electrophoresis of amplified YAC material. Ten microliters of PCR product was separated through 1.5% agarose (BRL) in 1X TAE (40 n&f Tris-acetate, 1 mA4 EDTA) containing 0.1 rg/ml ethidium bromide. Following destaining, DNA was visualized and photographed under ultraviolet light. Molecular weight standard is a 123-bp DNA ladder (BRL). Samples are RSll, RS46, and RS47, which map by Alu-PCR to Xq27.

ligation products

inserts. The primer directs

YACadapt oligonucleotide with a &n&AI cohePCR amplification of ligation products between

Repeat sequences were quenched by preassociating the denatured probe to 1 mg of sonicated human placental DNA for 7 min at 65°C essentially as described (9). A radiolabeled probe from YAC clone RS46 detects EcoRI fragments ranging from 14.5 to 2.5 kb and totalling 46.6 kb from the X chromosome. The entire YAC is relatively small, being 80 kb by pulsed-field hybridization with an insert of approximately 70.4 kb. Thus, the PCR products detect -70% of the human DNA present in this clone. Similar bands are detected in hybrids containing the entire X and Xpter-q27.3 (16), but are absent from those containing Xpter-q27.1 (4) and Xq28 (17), mapping YAC RS46 in the Xq27.2-27.3 region. Also of interest is the observation of conserved sequences present in the insert as demonstrated by a strongly hybridizing rodent band. Such cross-hybridization may be indicative of an expressed sequence present within this YAC. In addition, autosomal fragments are detected in a hybrid known to contain other human chromosomes [lane 71 (4), which is likely due to insert homology since YAC RS46 appears not to be chimeric. Results similar to these have been seen with other YAC clones. To determine if the PCR product was suitable for other physical mapping techniques, in situ hybridization was performed. Two hundred nanograms of Centriconpurified PCR product was labeled with biotinylated dUTP by nick-translation. The purified probe was hybridized, with repeat suppression, to metaphase chromosome preparations of the intact X hybrid Y751BMl

a

xq273

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xq273

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xq273

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xq273

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xq273

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xq273

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xq213

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9.4 kb -human c rodent +

human

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+23kb

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FIG. 3. Physical mapping of YAC RS46. (a) Hybridization of PCR-amplified RS46 material to a somatic cell hybrid mapping panel. Genomic DNAs were digested with EcoRI, separated on a 0.8% agarose gel in 1X TAE (40 mM Tris-acetate, 1 r&f EDTA), and transferred to Zetaprobe (Bio-Rad) nylon membrane by standard procedures. One hundred nanograms of PCR product was labeled by nicktranslation, purified, and hybridized with repeat suppression to the Xq deletion filter in 0.25 M NaPO,, 0.25 M NaCl, 7% (w/v) sodium dodecyl sulfate, 10% (w/v) polyethylene glycol, 1% (w/v) bovine serum albumin for 18 h. Washing was performed with stringencies starting at 2X SSC (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate) and ending with 0.15~ SSC at 65°C. The filter was exposed to Kodak XRP film for 5 days at -80°C with intensifying screens. Samples are normal male (lanes 1, 2), fragile X male (lanes 3, 4), hamster (lane 5), intact X-only microcell hybrid Y751BMl (lane 6), Xpter-q27.1 hybrid (lane 7), Xpter-q27.3 microcell hybrid (lane 8), Xq28-only hybrids (lanes 9-12). (b) In situ hybridization of RS46 PCR product to metaphase chromosome preparations. Two hundred nanograms of PCR product was labeled with biotinylated dUTP and hybridized with repeat suppression to metaphase chromosome spreads of the X-only microcell hybrid Y751BMl. Two dots corresponding to RS46 hybridization to each chromatid are visible in the general vicinity of Xq27.3.

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essentially as described (6). Hybridization was detected using avidin conjugated to fluorescein (FITC) and amplified with goat anti-avidin-FITC and a second incubation with avidin-FITC. For analysis, chromosomes were stained with propidium iodide in antifade medium and observed under fluorescent microscopy using an FITC filter. Figure 3b shows localization of the YAC to distal Xq, consistent with the deletion mapping described above. We have reported a novel strategy for the physical localization of yeast artificial chromosome clones to specific regions of the genome. This strategy has been employed to identify a YAC that maps to the area in distal Xq known to contain the fragile X site and was subsequently instrumental in cloning the fragile site and the associated gene FMR-l(14). As a PCR-based technique, this procedure provides a virtually limitless supply of DNA. In contrast, strategies designed to use the isolated YAC as probe (15) are hindered by the relatively small amount of DNA recovered from pulsed-field gels. The PCR amplification approach described complements most other strategies currently in use to analyze and order YAC clones: vector-AZu PCR (5), inverse PCR from vector end primers (lo), plasmid recovery of insert ends (13), and interspersed repetitive sequence PCR (4). However, none of these approaches provides any global analysis of the YAC itself. Comprehensive PCR recovery in this manner has the potential for representing most of the genomic DNA present in a given clone, allowing the direct identification of genes whose exons are present within a YAC by hybridization to cDNA libraries.

5.

C. T. (1989). Ah polymerase chain reaction: A method for rapid isolation of human specific sequences from complex DNA sources. Proc. Natl. Acad. Sci. USA 86: 6686-6690. Nelson, D. L., Ballabio, A., Victoria, M. F., Pieretti, M., Bies, R. D., Gibbs, R. A., Maley, J. A., Chinault, A. C., Webster, T. D., and Caskey, C. T. (1991). ALU-primed polymerase chain reaction for regional assignment of 110 yeast artificial chromosome clones from the human X chromosome: Identification of clones associated with a disease locus. Proc. Natl. Acad. Sci. USA 88: 6157-6161.

6.

Pinkel, analysis ization.

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Rigby, P. W. J., Dieckmann, M., Rhodes, C., andBerg, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113: 237-251.

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P. G., Whittaker, P. A., and Southern, E. M. (1985). Reof repeated sequences from hybridisation probes. Nucleic Acids Res. 13: 1905-1922. Silverman, G. A., Ye, R. D., Pollock, K. M., Sadler, J. E., and Korsmeyer, S. J. (1989). Use of yeast artificial chromosomes clones for mapping and walking within human chromosome segment 18q21.3. Proc. Natl. Acad. Sci. USA 86: 7485-7489. Smith, C. L., Lawrance, S. K., Gillespie, G. A., Cantor, C. R., Weissman, S. M., and Collins, F. S. (1987). Strategies for mapping and cloning macroregions of mammalian genomes. In “Methods in Enzymology” (M. Gottesman, Ed.), Vol. 151, pp. 461-489, Academic Press, New York. Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. Traver, C. N., Klapholz, S., Hyman, R. W., and Davis, R. W. (1989). Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences. Proc. Natl. Acad.

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ACKNOWLEDGMENTS S.T.W. Institute. National

and C.T.C. are investigators in the Howard This research was supported by Grant Institutes of Health (S.T.W.).

14. Hughes Medical HGo0038 from the

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