Plant Molecular Biology Reporter 13 (2) 1995
pages 156-163
Protocol
Cloning Genomic Sequences Using Long-Range PCR John Mundy, Raphael Mayer, and Nam-Hai Chua E-mail:
[email protected] (JM, RM, NHC) Laboratory of Plant Molecular Biology, Rockefeller University, 1230 York Avenue, New York, N.Y. 10021, USA; (JM) Department of Plant Physiology, Copenhagen University, Oster Farimagsgade 2A, 1353 Copenhagen K, Denmark Key Words: Arabidopsis thaliana, gene, gibberellic acid, inverse PCR, Iambda phage, promoter Abstract: Protocols are presented for preparing DNA from a genomic library in phage and for synthesizing genomic fragments using PCR with nested vectorand gene-specific primers and linker-primers. Library DNA, isolated from E. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The isolation of 5' upstream sequences of three different arabidopsis genes by this method is described. he isolation and sequencing of genomic clones, often part of the characterization of genes of interest, yields information on their structure and provides DNA fragments for studies of gene regulation. Genomic isolation is performed in many laboratories by DNA hybridization to genomic libraries in ~ phage vectors. While routine, this method involves several steps including library screening, and the isolation, restriction mapping and subcloning of phage DNA (Sambrook et al., 1989).
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Abbreviations: LB, Luria-Bertani medium; PCR, polymerase chain reaction; PEG, polyethylene glycol; TAE, tris-acetate buffer; UTR, untranslated sequence.
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PCR-based protocols incorporating recent improvements in synthetic fidelity and range offer easier and more rapid alternatives (Barnes, 1994; Chen et al., 1994). Thus, inverse PCR can be used to isolate fragments directly from preparations of genomic DNA without the need for genomic libraries (Ochman et al., 1990). The use of DNA from a representative genomic library as a template in anchored PCR, however, may be desirable for several reasons. 9 Library DNA can be reproducibly prepared in large amounts in many laboratories. 9 Vectors may contain polylinker restriction sites that simplify directional product subcloning. 9 Depending upon the method of library construction, multiple PCR products may be obtained in a single reaction representing 5- or 3'deletion series. These potential advantages prompted us to develop protocols for cloning specific, linker-containing genomic fragments from a representative and available genomic library of Arabidopsis thaliana ecotype Columbia. Due to our interest in phytohormone-regulated gene expression, we chose to isolate 5'-upstream sequences from three genes reported to be responsive to gibberellic acid: GAS1 (Shi et al., 1992; Herzog et al., 1995), TIP1 (H6 fte et al., 1992; Phillips & Huttly, 1994), and BIB1 (Phillips and Huttly, 1994). The same general protocols can be applied to the isolation of cDNA and genomic sequences from other ~ phage libraries.
Materials and Methods Isolation of library DNA 9 Inoculate: mix well 15 mL of KW251 cells (Promega, grown overnight in LB with 10 mM MgSO 4,0.2 % maltose) with 1.6xl 08pfu (300 ~tL GEM11 library1), incubate 15 min at room temperature. 9 Amplify: add 165 mL LB with 5 mM CaCI2, shake hard in three 100mL erlenmeyer flasks 4 to 5 hr at 37 ~ until lysed (a non-inoculated control may be useful). 9 Clear lysate: add 1 mL chloroform/flask, shake 5 min, transfer to Oak Ridge tubes, and centrifuge 4000 rpm, 5 min. 9 Precipitate phage: transfer aqueous phase to corex tubes, add equal volume of 20 % PEG 6000, 2M NaC1, mix well, incubate on ice I hr. 9 Collect phage: centrifuge at 6000 rpm, 20 min, 4 ~ discard supernatant and wipe tube side dry.
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9 Remove contaminants: resuspend pellets in 30 mL LB, add 30 mL DE52/LB, 2 mix by inversion 5 min. Centrifuge 5000 rpm, 5 min, transfer supernatant, repeat centrifugation and transfer to remove all DE52. 9 Disrupt phage: add 500 ~tL 0.1 m g / m L Proteinase K, incubate 30 rain. 37 ~ Add 1 mL 10 % SDS to supernatant, mix well and incubate 5 min at room temperature. Add 4 mL 3 M potassium acetate, incubate 20 min at 88 ~ cool on ice 10 min, centrifuge 5000 rpm, 10 rain, 4 ~ 9 Precipitate DNA: Transfer supernatant, add an equalvol, of isopropanol, incubate 20 min at -70 ~ Centrifuge 5000 rpm, 15 min at 4 ~ Discard supernatant, wash pellet with 10 mL 70 % EtOH, centrifuge 5 min, discard supernatant, and dry pellet briefly under vacuum. 9 Resuspend DNA: add 2 mL TE, shake gently overnight, store at 4 ~ or -20 ~ 9 Expected yield: approximately 200 to 300 ~tg K library DNA. Notes: 1. This genomic library of Arabidopsis thaliana, ecotype Columbia DNA was constructed by John T. Mulligan and Ronald W. Davis (unpublished). It contains approximately 20 kb, randomly sheared DNA fragments that were blunt ended, ligated to Eco RI adaptors, and cloned in X GEM11 (Promega #B1510). Aliquots can be obtained from: R.W. Davis or EEC-BRIDGEArabidopsis DNA Stock Center Attn. Marj Thomas Max-Delbruck Laboratory Dept. of Biochemistry Max-Planck Institute Stanford University Carl-von-Linn6 -Weg 10 Stanford CA 94305 D-50829 Cologne U.S.A. Germany 2. Wash 50 g DE-52 cellulose (Whatman) with 0.05 N HC1 to pH
