recovery of the selected cDNAs by the PCR. The procedure .... Nylon Discs and Hybridization for Selection of cDNAs. Non- .... on nylon discs (data not shown).
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*, SHERMAN M. WEISSMAN*
AND
*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 fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus.
Large segments of DNA from complex genomes are becoming available as yeast artificial chromosomes (YACs) (1, 2), cosmid or phage contigs (3, 4), fragments enriched by affinity capture (5), or chromosomal segments isolated in a foreign background in somatic cell hybrids (6, 7). Current estimates are that only a few percent of total animal cell DNA is a template for mRNA and that 10-20% of all mRNAs may be expressed in any particular cell type. A large part of the information content of the DNA that is of biologic relevance for the organism could be extracted from the cDNA sequences. Methods to identify mRNA templates include the identification of sequences conserved between divergent species such as man and mouse (8-11), use of these sequences as probes for hybridization to cDNA libraries, direct screening of cDNA libraries with probes made from the entire large fragment under conditions that suppress hybridization due to repetitive sequences (12), and analysis of sequences around CpG-rich islands that lie at the 5' ends of many but not all mRNA templates (13, 14). More recently PCR procedures with consensus primers for Alu repetitive sequences in heterogeneous nuclear RNA (15, 16) and exon trapping methods that depend on in vivo splicing systems to detect DNA sequences encoding RNA splicing sites (17, 30) have been introduced. We report here a conceptually simple alternative approach, cDNA selection, that is experimentally rapid and relatively insensitive to the size of the DNA fragments to be screened.
MATERIALS AND METHODS Preparation of Random Short-Fragment cDNA Library. A human spleen cDNA library was prepared using random hexamer primers as described (18). Preparation of Genomic Target DNA-Cosmids and YACs. Cosmid DNA was prepared by standard methods (19). YAC 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.
B30H3, isolated by one of us (D.D.C.; ref. 20), encompasses a 320-kilobase (kb) region from the human major histocompatibility complex (MHC) class I HLA-A locus. Yeast chromosomes from agarose plugs were fractionated by 1% agarose contour-clamped homogeneous electric field gel electrophoresis (21) in 0.5x TBE (45 mM Tris HCl, pH 8.3/45 mM boric acid/0.2 mM EDTA) at 14'C for 45 hr at 150 V/cm with a switching interval of 30 sec in an LKB Pulsaphor apparatus. A 320-kb YAC B30H3 band was excised and digested in situ with EcoPJ, and DNA was electroeluted. Immobilization of Genomic Cosmid or YAC DNA on Nylon Discs. Cosmid DNA (0.2 ng) or YAC DNA (1-2 ng) was digested with EcoPJ, heat-denatured at 950C for 3 min along with 50 ng of an Hae III digest of bacteriophage 4X174 DNA, as carrier DNA, and loaded in a 0.5-,ul volume onto 6.25-mm2 nylon discs (Hybond, Amersham), in the presence of 1Ox SSC (lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0). The DNA on discs was denatured in 0.5 M NaOH containing 1.5 M NaCl for 5 min. The discs were neutralized with 0.5 M Tris Cl (pH 7.2) containing 1.5 M NaCl for 3 min, either exposed to UV irradiation in a Stratalinker (Stratagene) for 1 min (autolink mode) or baked at 80'C in vacuum for 2 hr, and stored dry at 40C until used. Quenchers of Nonspecific Hybridization to Nylon Discs. (i) Human chromosome 15 library. DNA from a human chromosome 15 genomic library (15 NSO3, ATCC 57740) was digested with EcoRI and 0.8- to 6-kb (size range) DNA was cloned into the pTZ18 vector (Pharmacia) at the EcoRI site. A total of 3 x 105 recombinants was generated after transformation and ampicillin selection. (ii) Genomic repetitive sequence library (XRLI). XRLI was prepared from the human X chromosome library (LAOXNLO1, ATTC 57750) by digestion of various DNA samples with several single restriction enzymes, pooling, cloning 0.3- to 2-kb (size fraction) DNA in Charon BS vector (22), and probing the resulting library with 32P-labeled total genomic DNA (23). Five hundred positive plaques were processed for isolation of phage DNA and subsequent plasmid preparation (22). (iii) rRNA-specific clones. Plasmid clones containing human rRNA gene EcoRI fragments of 7.3 kb and 5.8 kb cloned into pBR322 at the EcoRI site were kindly provided by D. Ward (Yale University) (24). (iv) Poly(dI)-poly(dC). Material of 724 base pairs (average length; Pharmacia) was used without any further treatment. (v) Yeast DNA. Yeast DNA from strain AB1380, which is the parent host of YAC B30H3, was prepared (20) and sonicated to an average size of 0.7-1 kb. Plasmid DNA from the sources i-iii was sonicated, digested with EcoRI, and treated with 1 unit of mung bean nuclease per pug of DNA at Abbreviations: MHC, major histocompatibility complex; PFGE,
pulsed-field gel electrophoresis; YAC, yeast artificial chromosome. TTo whom reprint requests should be addressed.
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Genetics: Parimoo et al.
room temperature for 30 min to generate blunt-ended fragments of DNA ranging from 0.3 to 1 kb in size. Conditions for Quenching of Nonspecific Bindi"g of DNA to Nylon Discs and Hybridization for Selection of cDNAs. Nonspecific binding to target DNA was suppressed by incubating 6.25-mm2 nylon discs with immobilized DNA at 65TC in 40 ILI of solution containing 5x SSPE (lx SSPE = 0.15 M NaCI/ 0.02 M sodium phosphate/i mM EDTA, pH 7.2), 5x Denhardt's solution (19), 0.5% SDS, and a mixture of quenching agents described above, after heat denaturation at 950C for 5 min. The final concentrations of quenching agents in the reaction mixture were as follows: for XRLI, 0.025 ug/jl~; for chromosome 15 DNA fragments, 0.05 jxg/Al; for rRNA plasmid, 0.04 Ag/,ul; for poly(dI)-poly(dC), 0.02 ,ug/Al; and for sonicated yeast DNA (for YACs only), 0.025 ,ugId. After incubation under mineral oil for 20-24 hr, discs were briefly rinsed with 5x SSPE/0.1% SDS at room temperature and quickly transferred to a fresh tube containing 30 ul4 of hybridization solution of the same composition as the quenching reaction except that poly(dI)-poly(dC) was replaced by a heat-denatured short-fragment cDNA library (PCR-amplified with primer set C, defined below) at a concentration of 10 pug/ml. After an incubation of 36-40 hr at 65°C under mineral oil, the discs were washed for two 5-min periods with 2x SSC/0.1% SDS at room temperature, for three 20-min periods with 2x SSC/0.1% SDS at 650C, for one 20-min period with lx SSC/0.1% SDS at 65°C, for one 10-min period with 0.2x SSC/0.1% SDS at 650C, and for two 20-min periods with O.1x SSC/0.1% SDS at 65°C in 1.5-ml Eppendorf tubes with 6001l of wash solution. The discs were transferred to fresh tubes and rinsed twice with 0.lx SSC alone at room temperature. The discs were kept wet throughout the procedure and were either used immediately for the PCR or stored in 20 ,ul of water at 4°C overnight and then the PCR was carried out in the same tube. PCR Amplification. Two sets of primers, sets A and B, have been described earlier as primer sets 2 and 1, respectively (18). Another set of primers, set C, is composed of primer C1 (5'-CCACCTTTTGAGCAAGTTCAG-3') and primer C2 (5'-GAGGTGGCTTAT GAGTATTTC-3'), 18 and 20 bases away, respectively, from the EcoRI site of the AgtlO vector. Short-fragment cDNA inserts were liberated from a AgtlO random cDNA library by 30 cycles of PCR amplification in 100 ,ul, essentially as described (18), except that 2 mM MgCl2 and 0.9 ,M primer set C were used, and the reactions were carried out first by initial denaturation at 94°C for 2 min with all the components of the PCR mixture except AmpliTaq DNA polymerase (Cetus), followed by addition of the enzyme at 80°C. PCR cycles were 94°C for 70 sec, 50°C for 90 sec, and 72°C for 120 sec. PCR amplification of selected material on nylon discs was carried out by direct immersion of nylon disc in the PCR mixture and carrying out the PCR for 30 cycles as described for cDNA library amplification except that the primer (set C) concentration was reduced to 0.3 ,uM. A sample (1.5-2 1.l) from this PCR product was taken for a second round of 30 cycles of the PCR in the prqsence of the inner primer pair (set A). DNA Blots and Probes. Southern blot analysis of PCR products was done as described (18). The probes used for Southern blots were HLA-A cDNA (isolated by one of us-H.S.), MHC class II DRa (3-kb EcoRI fragment) (25), y-globin 200-base-pair fragment derived by PCR amplification of a globin cosmid (26), and total genomic DNA (sonicated material). Other probes have been described (18). Cloning of PCR-Amplifled and -Selected Material. A pool of five 100- / tubes of PCR reaction products from the second round of 30 cycles of PCR was concentrated on a Centricon100 (Amicon) by following the manufacturer's instructions and digested with EcoRI, and the cDNA material greater than
Proc. Natl. Acad. Sci. USA 88 (1991)
300 base pairs was cloned into phosphatase-treated AgtlO at the EcoRI site. The plaques were analyzed with the probes used for DNA blots.
RESULTS The basic protocol of selection is outlined in Fig. 1. The nylon filter discs with immobilized cosmid or YAC DNA were blocked for repetitive, ribosomal, or GC-rich sequences with quenching agents and then hybridized with total random cDNA fragments prepared by PCR amplification with flanking vector primers. After an appropriate wash, the hybridselected material was PCR-amplified, analyzed on Southern blots, and cloned in AgtlO. The first essential step for the cDNA selection was to establish methods and reagents that would reduce the coselection of repetitive and GC-rich sequences to a minimum. Hybridization Conditions for cDNA Selection. We investigated a number of conditions for hybridization and washing of nylon discs. Hybridization in SSPE, rather than in formamide, and washing conditions as described in Materials and Methods proved to be optimal as far as yield and specificity of the selection process were concerned (data not shown). A more stringent wash of 2.4 M tetraethylammonium chloride/50 mM Tris HCl, pH 8/2 mM EDTA/0.1% SDS (27) at various temperatures (50-600C) was investigated and could be used at 550C where the yield is of a lesser concern-e.g., when using higher concentrations of target DNA immobilized on nylon discs (data not shown). Specificity and Sensitivity of the Selection Process. To avoid PCR artifacts while providing the desired overall amplification, three sets of nested primers, with set A closest to the EcoRI cloning site and set C farthest from the cloning site, were tested for PCR amplification. Primer set B (between sets A and C) gave smaller-sized PCR products, compared to primer sets A and C (data not shown). Hence we routinely used primer sets C and A for subsequent reamplification stages (Fig. 1). The data in Fig. 2 indicate that the selection process is quite sensitive and can detect a signal on a DNA IGenornic DNA on nyvncr disc
