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Efficient PCR Approach for the Selection of Cdnas Encoded In Proc. Nati. Acad. Sci. USA Vol. 88, pp. 9623-9627, November 1991 Genetics cDNA selection: Efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments (yeast artificial chromosomes/cosmids/major histocompatibility complex) SATISH PARIMOO*t, SANKHAVARAM R. PATANJALI*, HRIDAYABHIRANJAN SHUKLA*, DAVID D. CHAPLIN*, AND SHERMAN M. WEISSMAN* *Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510; and tDepartment of Internal Medicine and Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110 Contributed by Sherman M. Weissman, July 24, 1991 ABSTRACT Identification of coding segments in large B30H3, isolated by one of us (D.D.C.; ref. 20), encompasses fragments of genomic DNA is a recurrent problem in genome a 320-kilobase (kb) region from the human major histocom- mapping and positional cloning studies. We have developed a patibility complex (MHC) class I HLA-A locus. Yeast chro- rapid and efficient protocol to achieve this goal, based on mosomes from agarose plugs were fractionated by 1% aga- hybridization of cDNA fragments to immobilized DNA and rose contour-clamped homogeneous electric field gel elec- recovery of the selected cDNAs by the PCR. The procedure trophoresis (21) in 0.5x TBE (45 mM Tris HCl, pH 8.3/45 permits rapid cloning of cDNA fragments encoded by large mM boric acid/0.2 mM EDTA) at 14'C for 45 hr at 150 V/cm genomic DNA fragments, groups of yeast artificial chromo- with a switching interval of 30 sec in an LKB Pulsaphor somes, or cosmids and has the potential to directly enrich apparatus. A 320-kb YAC B30H3 band was excised and cDNAs encoded in chromosome segments. By this approach we digested in situ with EcoPJ, and DNA was electroeluted. have been able to identify several non-major histocompatibility Immobilization of Genomic Cosmid or YAC DNA on Nylon complex class I clones from a yeast artificial chromosome that Discs. Cosmid DNA (0.2 ng) or YAC DNA (1-2 ng) was includes the HLA-A locus. digested with EcoPJ, heat-denatured at 950C for 3 min along with 50 ng ofan Hae III digest ofbacteriophage 4X174 DNA, Large segments of DNA from complex genomes are becom- as carrier DNA, and loaded in a 0.5-,ul volume onto 6.25-mm2 ing available as yeast artificial chromosomes (YACs) (1, 2), nylon discs (Hybond, Amersham), in the presence of 1Ox cosmid or phage contigs (3, 4), fragments enriched by affinity SSC (lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH capture (5), or chromosomal segments isolated in a foreign 7.0). The DNA on discs was denatured in 0.5 M NaOH background in somatic cell hybrids (6, 7). Current estimates containing 1.5 M NaCl for 5 min. The discs were neutralized are that only a few percent of total animal cell DNA is a with 0.5 M Tris Cl (pH 7.2) containing 1.5 M NaCl for 3 min, template for mRNA and that 10-20% of all mRNAs may be either exposed to UV irradiation in a Stratalinker (Strata- expressed in any particular cell type. A large part of the gene) for 1 min (autolink mode) or baked at 80'C in vacuum information content of the DNA that is of biologic relevance for 2 hr, and stored dry at 40C until used. for the organism could be extracted from the cDNA se- Quenchers of Nonspecific Hybridization to Nylon Discs. (i) quences. Methods to identify mRNA templates include the Human chromosome 15 library. DNA from a human chro- identification of sequences conserved mosome 15 genomic library (15 NSO3, ATCC 57740) was between divergent digested with EcoRI and 0.8- to 6-kb (size range) DNA was species such as man and mouse (8-11), use of these se- cloned into the pTZ18 vector (Pharmacia) at the EcoRI site. quences as probes for hybridization to cDNA libraries, direct A total of 3 x 105 recombinants was generated after trans- screening ofcDNA libraries with probes made from the entire formation and ampicillin selection. large fragment under conditions that suppress hybridization (ii) Genomic repetitive sequence library (XRLI). XRLI was due to repetitive sequences (12), and analysis of sequences prepared from the human X chromosome library around CpG-rich islands that lie at the 5' ends ofmany but not (LAOXNLO1, ATTC 57750) by digestion of various DNA all mRNA templates (13, 14). More recently PCR procedures samples with several single restriction enzymes, pooling, with consensus primers for Alu repetitive sequences in het- cloning 0.3- to 2-kb (size fraction) DNA in Charon BS vector erogeneous nuclear RNA (15, 16) and exon trapping methods (22), and probing the resulting library with 32P-labeled total that depend on in vivo splicing systems to detect DNA genomic DNA (23). Five hundred positive plaques were sequences encoding RNA splicing sites (17, 30) have been processed for isolation of phage DNA and subsequent plas- introduced. We report here a conceptually simple alternative mid preparation (22). approach, cDNA selection, that is experimentally rapid and (iii) rRNA-specific clones. Plasmid clones containing hu- relatively insensitive to the size of the DNA fragments to be man rRNA gene EcoRI fragments of7.3 kb and 5.8 kb cloned screened. into pBR322 at the EcoRI site were kindly provided by D. Ward (Yale University) (24). MATERIALS AND METHODS (iv) Poly(dI)-poly(dC). Material of 724 base pairs (average length; Pharmacia) was used without any further treatment. Preparation of Random Short-Fragment cDNA Library. A (v) Yeast DNA. Yeast DNA from strain AB1380, which is human spleen cDNA library was prepared using random the parent host of YAC B30H3, was prepared (20) and hexamer primers as described (18). sonicated to an average size of 0.7-1 kb. Plasmid DNA from Preparation of Genomic Target DNA-Cosmids and YACs. the sources i-iii was sonicated, digested with EcoRI, and Cosmid DNA was prepared by standard methods (19). YAC treated with 1 unit of mung bean nuclease per pug of DNA at The publication costs of this article were defrayed in part by page charge Abbreviations: MHC, major histocompatibility complex; PFGE, payment. This article must therefore be hereby marked "advertisement" pulsed-field gel electrophoresis; YAC, yeast artificial chromosome. in accordance with 18 U.S.C. §1734 solely to indicate this fact. TTo whom reprint requests should be addressed. 9623 Downloaded by guest on September 24, 2021 %24 Genetics: Parimoo et al. Proc. Natl. Acad. Sci. USA 88 (1991) room temperature for 30 min to generate blunt-ended frag- 300 base pairs was cloned into phosphatase-treated AgtlO at ments of DNA ranging from 0.3 to 1 kb in size. the EcoRI site. The plaques were analyzed with the probes Conditions for Quenching of Nonspecific Bindi"g of DNA to used for DNA blots. Nylon Discs and Hybridization for Selection of cDNAs. Non- specific binding to target DNA was suppressed by incubating 6.25-mm2 nylon discs with immobilized DNA at 65TC in 40 ILI RESULTS of solution containing 5x SSPE (lx SSPE = 0.15 M NaCI/ The basic protocol ofselection is outlined in Fig. 1. The nylon 0.02 M sodium phosphate/i mM EDTA, pH 7.2), 5x Den- filter discs with immobilized cosmid or YAC DNA were hardt's solution (19), 0.5% SDS, and a mixture of quenching blocked for repetitive, ribosomal, or GC-rich sequences with agents described above, after heat denaturation at 950C for 5 quenching agents and then hybridized with total random min. The final concentrations of quenching agents in the cDNA fragments prepared by PCR amplification with flank- reaction mixture were as follows: for XRLI, 0.025 ug/jl~; for ing vector primers. After an appropriate wash, the hybrid- chromosome 15 DNA fragments, 0.05 jxg/Al; for rRNA selected material was PCR-amplified, analyzed on Southern plasmid, 0.04 Ag/,ul; for poly(dI)-poly(dC), 0.02 ,ug/Al; and blots, and cloned in AgtlO. The first essential step for the for sonicated yeast DNA (for YACs only), 0.025 ,ugId. After cDNA selection was to establish methods and reagents that incubation under mineral oil for 20-24 hr, discs were briefly would reduce the coselection of repetitive and GC-rich rinsed with 5x SSPE/0.1% SDS at room temperature and sequences to a minimum. quickly transferred to a fresh tube containing 30 ul4 of Hybridization Conditions for cDNA Selection. We investi- hybridization solution of the same composition as the gated a number of conditions for hybridization and washing quenching reaction except that poly(dI)-poly(dC) was re- of nylon discs. Hybridization in SSPE, rather than in form- placed by a heat-denatured short-fragment cDNA library amide, and washing conditions as described in Materials and (PCR-amplified with primer set C, defined below) at a con- Methods proved to be optimal as far as yield and specificity centration of 10 pug/ml. After an incubation of 36-40 hr at of the selection process were concerned (data not shown). A 65°C under mineral oil, the discs were washed for two 5-min more stringent wash of 2.4 M tetraethylammonium chlo- periods with 2x SSC/0.1% SDS at room temperature, for ride/50 mM Tris HCl, pH 8/2 mM EDTA/0.1% SDS (27) at three 20-min periods with 2x SSC/0.1% SDS at 650C, for one various temperatures (50-600C) was investigated and could 20-min period with lx SSC/0.1% SDS at 65°C, for one be used at 550C where the yield is of a lesser concern-e.g., 10-min period with 0.2x SSC/0.1% SDS at 650C, and for two when using higher concentrations oftarget DNA immobilized 20-min periods with O.1x SSC/0.1% SDS at 65°C in 1.5-ml on nylon discs (data not shown).
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