recombinant dna part e

Preface Recombinant DNA methods are powerful, revolutionary techniques for at least two reasons. First, they allow the isolation of single genes in large amounts from a pool of thousands or millions of genes. Second, the isolated genes or their regulatory regions can be modified at will and reintroduced into cells for expression at the RNA or protein levels. These attributes allow us to solve complex biological problems and to produce new and better products in the areas of health, agriculture, and industry. Volumes 153, 154, and 155 supplement Volumes 68, 100, and 101 of Methods in Enzymology. During the past few years, many new or im- proved recombinant DNA methods have appeared, and a number of them are included in these three new volumes. Volume 153 covers methods related to new vectors for cloning DNA and for expression of cloned genes. Volume 154 includes methods for cloning cDNA, identification of cloned genes and mapping of genes, chemical synthesis and analysis of oligodeoxynucleotides, site-specific mutagenesis, and protein engineer- ing. Volume 155 includes the description of several useful new restriction enzymes, details of rapid methods for DNA sequence analysis, and a num- ber of other useful methods. RAY Wu LAWRENCE GROSSMAN xiii Contributors to Volume 154 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. TOM ALBER (27), Institute of Molecular Bi- ology, University of Oregon, Eugene, Or- egon 97403 DANNY C. ALEXANDER (3), Calgene, Inc., Davis, California 95616 K. ARAI (1), Department of Molecular Biol- ogy, DNAX Research Institute of Molec- ular and Cellular Biology, Palo Alto, Cali- fornia 94304 S. L. BEAUCAGE (15), Department of Genet- ics, Stanford University, Stanford, Cali- fornia 94305 HELMUT BLOCKER (13), GBF (Gesellschaft far Biotechnologische Forschung mbH), D-3300 Braunschweig, Federal Republic of Germany JEF D. BOEKE (10), Department of Molecu- lar Biology and Genetics, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205 M. BROWNSTEIN (1), Laboratory of Molecu- lar Genetics, National Institute of Child Health and Human Development, Be- thesda, Maryland 20205 PAUL CARTER (20), Department of Biomole- cular Chemistry, Genentech, Inc., South San Francisco, California 94080 M. H. CARUTHERS (15), Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309 PIERRE CHAMBON (14), Laboratoire de Gdndtique Moldculaire, LGME/CNRS et U.184/INSERM, Institute de Chimie Biologique, Facultd de Mddecine, 67085 Strasbourg Cedex, France CHRISTOPHER COLECLOUGH (4), Basel Insti- tute for Immunology, CH-4005 Basel, Switzerland A. D. DARONE (15), Centocor, Inc., Mal- vern, Pennsylvania 19355 RONALD W. DAVIS (7), Department of Bio- chemistry, Stanford University School of Medicine, Stanford, California 94305 D. R. DODDS (15), Sepracor, Inc., Marlbor- ough, Massachusetts 01752 STEPHEN ELLEDGE (7), Department of Bio- chemistry, Stanford University School of Medicine, Stanford, California 94305 GERALD R. FINK (10), Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, and Massachusetts Institute for Technology, Cambridge, Massachusetts 02139 JOSEPH R. FIRCA (16), Pandex Laborato- ries, Inc., Mundelein, Illinois 60060 E. F. FISHER (15), AMGen, Inc., Thousand Oaks, California 91360 RONALD FRANK (13), GBF (Gesellschaftfiir Biotechnologische Forschung mbH), D-3300 Braunschweig, Federal Republic of Germany HANS-JOACHIM FRITZ (18), Max-Planck-ln- stitut fiir Biochemie, Abteilung Zellbiolo- gie, Am Klopferspitz 18, D-8033 Mar- tinsried bei Miinchen, Federal Republic of Germany MARK R. GRAY (8), Department of Biologi- cal Chemistry, Harvard Medical School, Boston, Massachusetts 02115 GISELA HEIDECKER (2), Section of Genet- ics, Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701 LEROY HOOD (16), Division of Biology, Cal- ifornia Institute of Technology, Pasa- dena, California 91125 SUZANNA J. HORVATH (16), Division of Bi- ology, California Institute of Technology, Pasadena, California 91125 P. C. HUANG (22), Department of Chemis- try, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205 ix X CONTRIBUTORS TO VOLUME 154 MICHAEL W. HUNKAPILLER (16), Applied Biosystems, Inc., Foster City, California 94404 TIM HUNKAPILLER (16), Division of Biol- ogy, California Institute of Technology, Pasadena, California 91125 MITTUR N. JAGADISH (12), Division of Pro- tein Chemistry, CSIRO, Parkville 3052, Victoria, Australia E. T. KAISER (25), Laboratory of Bioorganic Chemistry and Biochemistry, The Rockefeller University, New York, New York 10021 M. KAWAICHI (1), Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Be- thesda, Maryland 20205 PETER KOLLMAN (23), Department of Phar- maceutical Chemistry, University of Cali- fornia, San Francisco, San Francisco, California 94143 WILFRIED KRAMER (18), Max-Planck-Insti- tut fiir Biochemie, Abteilung Zellbiologie, Am Klopferspitz 18, D-8033 Martinsried bei Miinchen, Federal Republic of Ger- many THOMAS A. KUNKEL (19), National Insti- tute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709 F. LEE (1), Department of Molecular Biol- ogy, DNAX Research Institute of Molec- Mar and Cellular Biology, Palo Alto, Cali- fornia 94304 COREY LEVENSON (21), Department of Chemistry, Cetus Corporation, Emery- ville, California 94608 DAVID F. MARK (21), Department of Molec- Mar Biology, Cetus Corporation, Emery- ville, California 94608 M. MATTEUCCl (15), Genentech, Inc., South San Francisco, California 94112 HANS W. DJURHUUS MATTHES (14), La- boratoire de Gdndtique Moldculaire, LGME/CNRS et U.184/INSERM, Insti- tute de Chimie Biologique, Facult~ de M~decine, 67085 Strasbourg Cedex, France BRIAN W. MATTHEWS (27), Institute of Mo- lecular Biology, University of Oregon, Eugene, Oregon 97403 C. ROBERT MATTHEWS (26), Department of Chemistry, The Pennsylvania State Uni- versity, University Park, Pennsylvania 16802 GAlL P. MAZZARA (8), Applied Biotechnol- ogy, Inc., Cambridge, Massachusetts 02139 L. J. McBRIDE (15), Applied Biosystems, Foster City, California 94404 JOACHIM MESSING (2), Waksman Institute of Microbiology, Rutgers University, Pis- caraway, New Jersey 08854 ANDREAS MEYERHANS (13), GBF (Gesell- schaft far Biotechnologische Forschung mbH), Mascheroder Weg 1, D-3300 Braunschweig, Federal Republic of Ger- many C. GARRETT MIYADA (6), Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, Cal- ifornia 91010 GEORGES NATSOULIS (10), Department of Molecular Biology and Genetics, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205 JOHN D. NOTI (12), Molecular and Cell Biol- ogy, Triton Biosciences, Inc., Alameda, California 94501 H. OKAYAMA (1),' Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892 RICHARD PINE (22), Department of Molecu- lar and Cell Biology, The Rockefeller Uni- versity, New York, New York 10021 SANDOR PONGOR (24), Institute of Enzymol- ogy, Hungarian Academy of Sciences, pf 7 Budapest 1502, Hungary, and Boyce Thompson Institute, Cornell University, Ithaca, New York 14853 PRANHITHA REDDY (8), Department of Biol- ogy, Massachusetts Institute of Technol- ogy, Cambridge, Massachusetts 02139 A. A. REYES (5), Department of Molecular Genetics, Beckman Research Institute of CONTRIBUTORS TO VOLUME 154 Xi the City of Hope, Duarte, California 91010 JOHN D. ROBERTS (19), National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709 MICHAEL ROSBASH (8), Department of Biol- ogy, Brandeis University, Waltham, Mas- sachusetts 02254 KONRAD SCHWELLNUS (13), GBF (Gesell- schaft fiir Biotechnologische Forschung mbH), Mascheroder Weg 1, D-3300 Braunschweig, Federal Republic of Ger- many MICHAEL SMITH (17), Department of BiD- chemistry, University of British Colum- bia, Vancover, British Columbia MICHAEL SNYDER (7), Department of BiD- chemistry, Stanford University School of Medicine, Stanford, California 94305 Z. STABINSKY (15), Department of Chemis- try and Biochemistry, University of Colo- rado, Boulder, Colorado 80309 ADRIEN STAUB (14), Laboratoire de Gdn~ti- que Mol~culaire, LGME/CNRS et U.184/ INSERM, Institute de Chimie Biologique, Facultd de M~decine, 67085 Strasbourg Cedex, France DOUGLAS SWEETSER (7), Whitehead Insti- tute for Biomedical Research, Cam- bridge, Massachusetts 02142 ALADAR A. SZALAY (12), Boyce Thompson Institute for Plant Research, Cornell Uni- versity, Ithaca, New York 14853 J Y. TANG (15), Shanghai Institute of BiD- chemistry, Shanghai, Peoples Republic of China JOHN W. TAYLOR (25), Laboratory of Bioorganic Chemistry and Biochemistry, The Rockefeller University, New York, New York 10021 JOSHUA TRUEHEART (10), Whitehead Insti- tute for Biomedical Research, Cam- bridge, Massachusetts 02142, and Mas- sachusetts Institute of Technology, Cam- bridge, Massachusetts 02139 R. BRUCE WALLACE (5, 6), Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, Cal- ifornia 91010 ALICE WANG (21), Department of Molecu- lar Biology, Cetus Corporation, Emery- ville, California 94608 GEORGE M. WEINSTOCK (9), Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas 77225 T. YOKOTA (1), Department of Molecular Biology, DNAX Research Institute of Mo- lecular and Cellular Biology, Paid Alto, California 94304 RICHARD A. YOUNG (7), Whitehead Insti- tute for Biomedical Research, Cam- bridge, Massachusetts 02142, and De- partment of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 RICHARD A. ZAKOUR (19), Molecular and Applied Genetics Laboratory, Allied Cor- poration, Morristown, New Jersey 07960 MARK J. ZOLLER (17), Cold Spring Harbor Laboratory, Cold Spring Harbor. New York 11724 FRANS J. DE BRUIJN (ll), Max-Planck-lnsti- tut fiir Zi~chtungsforschung, Abteilung Schell, D-5000 KOln 30, Federal Republic of Germany W.F. VAN GUNST (23), Department of Physical Chemistry, University of Gro- ningen, 9747 AG Groningen, The Nether- lands [1] CONSTRUCTION AND SCREENING OF cDNA LIBRARIES 3 [1] High-Efficiency Cloning of Full-Length cDNA; Construction and Screening of cDNA Expression Libraries for Mammalian Cells By H. OKAYAMA, M. KAWAICHI, M. BROWNSTEIN, F. LEE, T. YOKOTA, and K. ARAI cDNA cloning constitutes one of the essential steps to isolate and characterize complex eukaryotic genes, and to express them in a wide variety of host cells. Without cloned cDNA, it is extremely difficult to define the introns and exons, the coding and noncoding sequences, and the transcriptional promoter and terminator of genes. Cloning of cDNA, however, is generally far more difficult than any other recombinant DNA work, requiring multiple sequential enzymatic reactions. It involves in vitro synthesis of a DNA copy of mRNA, its subsequent conversion to a duplex cDNA, and insertion into an appropriate prokaryotic vector. Due to the intrinsic difficulty of these reactions as well as the inefficiency of the cloning protocols devised, the yield of clones is low and many of clones are truncated.1 The cloning method developed by Okayama and Berg 2 circumvents many of these problems, and permits a high yield of full-length cDNA clones regardless of their size. 3-6 The method utilizes two specially engi- neered plasmid DNA fragments, "vector primer" and "linker DNA." In addition, several specific enzymes are used for efficient synthesis of a duplex DNA copy of mRNA and for efficient insertion of this DNA into a plasmid. Excellent yields of full-length clones and the unidirectional in- sertion of cDNA into the vector are the result. These features not only facilitate cloning and analysis but are also ideally suited for the expression of functional cDNA. To take full advantage of the features of this method, Okayama and A. Efstratiadis and L. Villa-Komaroff, in "Genetic Engineering" (J. K. Setlow and A. HoUaender, eds.), Vol. 1, p. 1. Plenum, New York, 1979. 2 H. Okayama and P. Berg, Mol. Cell. Biol. 1, 161 (1982). 3 D. H. Maclennan, C. J. Brandl, B. Korczak, and N. M. Green, Nature (London) 316, 696 (1985). 4 L. C. Kun, A. McClelland, and F. H. Ruddle, Cell 37, 95 (1984). 5 K. Shigesada, G. R. Stark, J. A. Maley, L. A. Niswander, and J. N. Davidson, Mol. Cell. Biol. 5, 1735 (1985). 6 S. M. Hollenberg, C. Weinberger, E. S. Ong, G. Cerelli, A. Oro, R. Lebo, E. B. Thomp- son, M. G. Rosenfeld, and R. M. Evans, Nature (London) 318, 635 (1985). Copyright © 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 154 All rights of reproduction in any form reserved. 4 METHODS FOR CLONING eDNA [1] Berg 7 have modified the original vector. The modified vector, pcD, has had SV40 transcriptional signals introduced into the vector primer and linker DNAs to promote efficient expression of inserted cDNAs in mam- malian cells. Construction of eDNA libraries in the pcD expression vector thus permits screening or selection of particular clones on the basis of their expressed function in mammalian cells, in addition to regular screen- ing with hybridization probes. Expression cloning has proven extremely powerful if appropriate functional assays or genetic complementation selection systems are avail- able. 8-14 In fact, Yokota et al. 11,12 and Lee et al. 13,14 have recently isolated full-length eDNA clones encoding mouse and human lymphokines with- out any prior knowledge of their chemical properties, relying entirely on transient expression assays using cultured mammalian cells. Similar mod- ifications have been made to promote the expression of cDNA in yeast, thereby permitting yeast mutant cells to be used as possible complementa- tion hosts.tS,16 In this chapter, we describe detailed procedures for the construction of full-length cDNA expression libraries and the screening of the libraries for particular clones based on their transient expression in mammalian cells. Methods for library transduction and screening based on stable expression are described in Vol. 151 of Methods in Enzymology. If ex- pression cloning is not envisioned, the original vector 2 or one described by others 17 can be used with slight modifications of the procedure de- scribed below. 7 H. Okayama and P. Berg, Mol. Cell. Biol. 2, 280 (1983). s D. H. Joly, H. Okayama, P. Berg, A. C. Esty, D. Filpula, P. Bohlen, G. G. Johnson, J. E. Shivery, T. Hunkapiller, and T. Friedmann, Proc. Natl. Sci. Acad. U.S.A. 80, 477 (1983). 9 D. Ayusawa, K. Takeishi, S. Kaneda, K. Shimizu, H. Koyama, and T. Seno, J. Biol. Chem. 259, 1436 (1984). 10 H. Okayama and P. Berg, Mol. Cell. Biol. 5, 1136 (1985). 11 T. Yokota, F. Lee, D. Rennick, C. Hall, N. Arai, T. Mosmann, G. Nabel, H. Cantor, and K. Aral, Proc. Natl. Acad. Sci. U.S.A. 81, 1070 (1985). 12 T. Yokota, N. Arai, F. Lee, D. Rennick, T. Mosmann, and K. Arai, Proc. Natl. Acad. Sci. U.S.A. 82, 68 (1985). 13 F. Lee, T. Yokota, T. Otsuka, L. Gemmell, N. Larson, L. Luh, K. Arai, and D. Rennick, Proc. Natl. Acad. Sci. U.S.A. 82, 4360 (1985). 14 F. Lee, T. Yokota, T. Otsuka, P. Meyerson, D. Villaret, R. Coffman, T. Mosmann, D. Rennick, N. Roehm, C. Smith, C. Zlotnick, and K. Arai, Proc. Natl. Acad. Sci. U.S.A. 83, 2061 (1986). 15 G. L. McKnight and B. C. McConaughy, Proc. Natl. Acad. Sci. U.S.A. 80, 4412 (1983). 16 A. Miyajima, N. Nakayama, I. Miyajima, N. Arai, H. Okayama, and K. Arai, Nucleic Acids Res. 12, 6639 (1984). 17 D. C. Alexander, T. D. McKnight, and B. G. Williams, Gene 31, 79 (1984). [1] CONSTRUCTION AND SCREENING OF cDNA LIBRARIES 5 Methods Clean, intact mRNA is prepared from cultured cells or tissue by the guanidine thiocyanate method 18 followed by two cycles of oligo(dT)- cellulose column chromatography. The purified mRNA is then reverse transcribed by the avian myeloblastosis enzyme in a reaction primed with the pcD-based vector primer, a plasmid DNA fragment that contains a poly(dT) tail at one end and a HindlII restriction site near the other end (Figs. 1 and 2). 7 The vector also contains the SV40 poly(A) addition signal downstream of the tail site as well as the pBR322 replication origin and the/3-1actamase gene. Reverse transcription results in the synthesis of a cDNA: mRNA hybrid covalently linked to the vector molecule (Fig. 3). This product is tailed with oligo(dC) at its 3' ends and digested with HindlII to release an oligo(dC) tail from the vector end and to create a HindlII cohesive end. The C-tailed cDNA : mRNA hybrid linked to the vector is cyclized by addition of DNA ligase and a pcD-based linker DNA an oligo(dG)-tailed DNA fragment with a HindlII cohesive end (this linker contains the SV40 early promoter and the late splice junctions) (Figs. 1 and 2). Finally, the RNA strand is converted to DNA by nick- translation repair catalyzed by Escherichia coli DNA polymerase I, RNase H, and DNA ligase. The end product, a closed circular cDNA recombinant, is transfected into a highly competent E. coli host to estab- lish a cDNA clone library. In the steps that have just been enumerated, double-stranded, full- length DNA copies of the original mRNAs are efficiently synthesized and inserted into the vector to form a functional composite gene with the protein coding sequence derived from the cDNA and the transcriptional and RNA processing signals from the SV40 genome. To screen for or select a particular clone on the basis of the function it encodes, the library is acutely transfected or stably transduced into cultured cells. Procedures for stable transduction are described in Chap. [32] of Vol. 151 of Methods in Enzymology. Preparation of mRNA Successful construction of full-length cDNA libraries depends heavily on the quality of the mRNA preparation. The use of intact, uncontami- nated mRNA is essential for generating full-length clones. Messenger RNA prepared by the guanidine thiocyanate method 18 satisfies the above 18 j. M. Chiigwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18, 5294 (1978). 6 METHODS FOR CLONING cDNA [1] (O.NI •ell Hiadlll .v, "'°2'2(' pBR~2 orl o I Hindlll Sal I i a#l Awll 10,9151 im junctioe ~ C/at ~ Xhol 10.7§ -7"/ ,o., Hindlll (0.71) FBR322 ori - /f ~.~ SV4Oon / ~" x~l ,,~'~ Pstl pcO-X X(©DNA) P8 ~ ~, ~ 4 "t ~1 & 8mmHI p~A FIG. I. Structure and component parts of the pcD vector and its precursor plasmids, pcDV1 and pLl. The principal elements of the pcD vector are a segment containing the SV40 replication origin and the early promoter joined to a segment containing the 19 S and 16 S SV40 late splice junctions (hatched area); the various cDNA inserts flanked by dG/dC and dA/dT stretches that connect them to the vector (solid black area); a segment containing the SV40 late polyadenylation signal [poly(A)] (stippled area); and the segment containing the pBR322/3-1actamase gene and the origin of replication (thin and open area), pcDVl and pL 1 provide the pcD-based vector primer and linker DNA, respectively. For the preparation of the vector primer and linker DNA, see Methods sections and Fig. 2. [1] CONSTRUCTION AND SCREENING OF cDNA LIBRARIES 7 ] VECTOR PRIMER I Hind]J1 (0.86) . Eco RI (0.758) AmpR(/// pcDVI "~ ,0.754) ~ (0.71).~ KpnI (0.715) \\ (0.14~!~ XhoI 8 BamHI ~~ "polylA) "(0.19l KpnI DIGESTION POLY (dT) TAILS • HindgI Eco RI ~~ Kpn I Eco RI DIGESTION PURIFICATION I OLIGO (dO) TAILED LINKER DNA ] (0.95) PstI 16S splice ~ junction Amp R // (0.93)~ ~ X'h^' {0.75) f/ PstI-~ pL] ~ SV40 ori ~~~./ "Hind l]] (0.71) ~ PstI DIGESTION OLIGO (dG) TAILS / ~ G G G ~~ /\ Hind HI Hindl]I DIGESTION PURIFICATION Hind ]]] ~T ECO RI Xho I TTTT Pstl G Xho I ~ Bam HI ~a FIG. 2. Preparation of vector primer and linker DNAs. Hind In criteria and is reverse transcribed efficiently. It has successfully been used for cloning a number of cDNAs? -14 The method described below is a slight modification of the original method that ensures complete inactiva- tion of RNases through all the steps of RNA isolation. 8 METHODS FOR CLONING cDNA [1] mRNA ~ ANNEALING: ~. Cc c .~ cDNA ~ - OLIGO SYNT.ES,S TA,LS A ,- % ~ 'T~ ,,c _ [ f T ~ /~ TT.A - llff~ T k Hindlll Hindlll~Xh°l ~ BamHl ~ PRIMER AC C C Ps, I f2 t "~ "~-AA /H~m H/ndlll ~GG G ¢/~ , ~- -T T A J DIGESTION ANNEALING OF OLIGO dG ' Hindm Xho[ 8BamHl TAILED LINKER; CYCLIZATION WITH E.co//DNA LIGASE / ~.G~ ~ ~ __~- REPLACEMENT OF RNA STRAND GGc~'~ ~ DNA POLYMERASE 1 AND "~" tl~ A "~ ~ ~:~ DNA UGASE Hk'~dH] 'P~ TAA H/ndln FIG. 3. Enzymatic steps in the construction of pcD-cDNA recombinants. The designa- tions of the DNA segments are as described in Fig. 1. For experimental details and com- ments, see Methods. Reagents All solutions are prepared using autoclaved glassware or sterile dis- posable plasticware, autoclaved double-distilled water and chemicals of the finest grade. Solutions are sterilized by filtration through Nalgen 0.45 /zm Millipore filters and subsequently by autoclaving (except as noted). In general, treatment of solutions with diethyl pyrocarbonate is not recom- mended since residual diethyl pyrocarbonate may modify the RNA, re- sulting in a marked reduction in its template activity. 5.5 M GTC solution: 5.5 M guanidine thiocyanate (Fluka or Eastman-Kodak), 25 mM sodium citrate, 0.5% sodium lauryl sarcosine. After the pH is ad- justed to 7.0 with NaOH, the solution is filter-sterilized and stored at 4 °. Prior to use, 2-mercaptoethanol is added to a final concentra- tion of 0.2 M. 4 M GTC solution: 5.5 M solution diluted to 4 M with sterile distilled water. CsTFA solution: cesium trifluoroacetate (density 1.51 - 0.01 g/ml), 0.1 M ethylene- diaminetetraacetic acid (EDTA) (pH 7.0). Prepared with cesium trifluoroacetate (2 g/ml) (CsTFA, Pharmacia) and 0.25 M EDTA (pH 7.0). [...]... hybridization experiments 6,7 Various methods for the cloning of cDNA have been developed since the discovery of reverse transcriptase, the enzyme that made it all possible 8-~° The method we present here combines high efficiency with relative simplicity Principles The principal steps of our cDNA cloning procedure are outlined in Fig 1 The 3' ends of a linearized pUC plasmid are extended with thymidine residues... to ensure complete removal of free deoxynucleoside triphosphates Finally the pellet is rinsed with ethanol prior to the next step The yield of product after three ethanol precipitations is 70-80% Comments The use of clean, intact mRNA, fresh RNase-free reverse transcriptase, and a well-prepared vector primer is essential for the efficient synthesis of long cDNA Inclusion of RNase inhibitors in the reaction... one needs an appropriate recipient cell line Because the pcD vector carries the SV40 early promoter and origin of replication, COS cells have been used as hosts These cells contain an origin-defective SV40 genome and constitutively produce T antigen, a viral gene product needed to direct DNA replication initiating at the SV40 origin 25 These cells are capable of greatly amplifying the number of DNA. .. molecules taken up by the trans25 y Gluzman, Cell 23, 175 (1981) 26 METHODSFOR CLONINGcDNA [1] fected cells, increasing the amount of gene product synthesized The second requirement is an efficient method for introducing DNA into the COS cells Based on previous studies, DEAE-dextran has been used to effectively introduce DNA into recipient cells Immediately after introducing DNA into the cells they... (Pharmacia), nuclease free Contamination of the E coli enzyme preparations by endonuclease specific for double- or single-stranded DNA can be detected by digestion of supercoiled pBR322 or single-stranded ~bX174 DNAs under the conditions specified below for each enzyme followed by analysis of their degradation by agarose gel electrophoresis Procedure One microliter of the HindlII-digested, C-tailed m R N A... as primer for synthesis of the second strand of the cDNA insert after which the recombinant plasmids are transfected into Escherichia coli Experimental Procedures Materials All restriction enzymes were purchased from either New England Biolabs or Bethesda Research Laboratories (BRL) MLV reverse transcriptase, placental RNase inhibitor (RNasin), and large fragment of E coli DNA polymerase I were also... cDNAs are very useful in several regards Even short clones provide molecular probes, the tools to facilitate the isolation of homologous genomic clones and additional cDNA clones The sequence of fulllength cDNA clones allows one to deduce the amino acid sequence of the encoded protein 5 Full-length cDNA clones have been invaluable in the analysis of the organization and regulation of eukaryotic genes in... diameter × 2.5 cm height) The column is washed with several bed volumes of sterile distilled water and equilibrated with the loading buffer at 0-4 ° The DNA pellet is dissolved in 1 ml of the loading buffer, cooled on ice and applied to the column After the column is washed with several bed volumes of the buffer at 04 °, the bound DNA is eluted with sterile distilled water at room temperature One milliliter... 15° After centrifugation, the upper GTC layer and the DNA band at the interface are removed by aspiration The tubes are quickly inverted, and their contents are poured into a beaker Still inverted, they are placed on a paper towel to drain for 5 min, and then the bottom 2 cm of the tube is cut off with a razor blade or scalpel; the remainder is discarded After the bottom of the tube is removed, the cup... approach does not require any prior knowledge of the protein product itself One only needs a specific biological or enzymatic assay for the presence of the protein in the cells or medium For the techniques to work, the activity sought must be attributable to a single gene product In addition to the library of cDNA clones to be transfected, there are two components necessary for transient expression screening . oligodeoxynucleotides, site-specific mutagenesis, and protein engineer- ing. Volume 155 includes the description of several useful new restriction enzymes, details of rapid methods for DNA sequence. dissociate the DNA. The TE is separated from the beads by brief centrifugation in a microfuge; then the beads are extracted once more with 1 ml of TE. Both extracts are pooled, and, after several. chapter, we describe detailed procedures for the construction of full-length cDNA expression libraries and the screening of the libraries for particular clones based on their transient expression