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 551 supplement Volumes 68, 100, and 101 of Methods ni 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 351 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 551 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. YAR Wu ECNERWAL NAMSSORG iiix Contributors to Volume 154 elcitrA srebmun are ni sesehtnerap gniwollof the seman of .srotubirtnoc snoitailiffA listed era .tnerruc TOM ALBER (27), Institute of Molecular Bi- Medicine, Stanford, California 50349 ology, University of Oregon, Eugene, Or- D. R. DODDS (15), Sepracor, Inc., Marlbor- egon 30479 ough, Massachusetts 25710 DANNY C. ALEXANDER (3), Calgene, Inc., STEPHEN ELLEDGE (7), Department of Bio- Davis, California 95616 chemistry, Stanford University School of K. ARAI (1), Department of Molecular Biol- Medicine, Stanford, California 50349 ogy, DNAX Research Institute of Molec- GERALD R. FINK (10), Whitehead Institute ular and Cellular Biology, Palo Alto, Cali- for Biomedical Research, Cambridge, fornia 40349 Massachusetts 02142, and Massachusetts S. L. BEAUCAGE (15), Department of Genet- Institute for Technology, Cambridge, ics, Stanford University, Stanford, Cali- Massachusetts 02139 fornia 50349 JOSEPH R. FIRCA (16), Pandex Laborato- HELMUT BLOCKER (13), GBF (Gesellschaft ries, Inc., Mundelein, Illinois 60060 far Biotechnologische Forschung mbH), D-3300 Braunschweig, Federal Republic E. F. FISHER (15), AMGen, Inc., Thousand Oaks, California 91360 of Germany JEF D. BOEKE (10), Department of Molecu- RONALD FRANK (13), GBF (Gesellschaftfiir lar Biology and Genetics, The Johns Biotechnologische Forschung mbH), D-3300 Braunschweig, Federal Republic Hopkins University, School of Medicine, of Germany Baltimore, Maryland 50212 M. BROWNSTEIN (1), Laboratory of Molecu- HANS-JOACHIM FRITZ (18), Max-Planck-ln- lar Genetics, National Institute of Child stitut fiir Biochemie, Abteilung Zellbiolo- Health and Human Development, Be- gie, Am Klopferspitz ,81 D-8033 Mar- thesda, Maryland 50202 tinsried bei Miinchen, Federal Republic of Germany PAUL CARTER (20), Department of Biomole- cular Chemistry, Genentech, Inc., South MARK R. GRAY (8), Department of Biologi- San Francisco, California 08049 cal Chemistry, Harvard Medical School, Boston, Massachusetts 51120 M. H. CARUTHERS (15), Department of Chemistry and Biochemistry, University GISELA HEIDECKER (2), Section of Genet- of Colorado, Boulder, Colorado 80309 ics, Laboratory of Viral Carcinogenesis, PIERRE CHAMBON (14), Laboratoire de National Cancer Institute, Frederick, Gdndtique Moldculaire, LGME/CNRS et Maryland 10712 U.184/INSERM, Institute de Chimie LEROY HOOD (16), Division of Biology, Cal- Biologique, Facultd de Mddecine, 67085 ifornia Institute of Technology, Pasa- Strasbourg Cedex, France dena, California 52119 COLECLOUGH CHRISTOPHER (4), Basel Insti- SUZANNA J. HORVATH (16), Division of Bi- tute for Immunology, CH-4005 Basel, ology, California Institute of Technology, Switzerland Pasadena, California 52119 A. D. DARONE (15), Centocor, Inc., Mal- P. C. HUANG (22), Department of Chemis- vern, Pennsylvania 55391 try, The Johns Hopkins University, RONALD W. DAVIS (7), Department of Bio- School of Hygiene and Public Health, chemistry, Stanford University School of Baltimore, Maryland 50212 ix X CONTRIBUTORS TO VOLUME 154 MICHAEL W. (16), HUNKAPILLER Applied BRIAN W. SWEHTTAM (27), Institute of Mo- Biosystems, Inc., Foster City, California lecular Biology, University of Oregon, 40449 Eugene, Oregon 30479 TIM (16), HUNKAPILLER Division of Biol- C. (26), MATTHEWS ROBERT Department of ogy, California Institute of Technology, Chemistry, The Pennsylvania State Uni- Pasadena, California 91125 versity, University Park, Pennsylvania MITTUR N. (12), JAGADISH Division of Pro- 20861 tein Chemistry, CSIRO, Parkville 3052, GAlL P. (8), MAZZARA Applied Biotechnol- Victoria, Australia ogy, Inc., Cambridge, Massachusetts E. T. KAISER (25), Laboratory of 93120 Bioorganic Chemistry and Biochemistry, L. J. McBRIDE (15), Applied Biosystems, The Rockefeller University, New York, Foster City, California 94404 New York 12001 MIHCAOJ (2), MESSING Waksman Institute M. (1), KAWAICHI Laboratory of Molecular of Microbiology, Rutgers University, Pis- Genetics, National Institute of Child caraway, New Jersey 08854 Health and Human Development, Be- SAERDNA SNAHREYEM (13), GBF (Gesell- thesda, Maryland 20205 schaft far Biotechnologische Forschung (23), KOLLMAN PETER Department of Phar- mbH), Mascheroder Weg ,1 D-3300 maceutical Chemistry, University of Cali- Braunschweig, Federal Republic of Ger- fornia, San Francisco, San Francisco, many California 34149 C. GARRETT MIYADA (6), Department of (18), WILFRIED KRAMER Max-Planck-Insti- Molecular Genetics, Beckman Research tut fiir Biochemie, Abteilung Zellbiologie, Institute of the City of Hope, Duarte, Cal- Am Klopferspitz ,81 D-8033 Martinsried ifornia 91010 bei Miinchen, Federal Republic of Ger- SEGROEG (10), NATSOULIS Department of many Molecular Biology and Genetics, The SAMOHT A. KUNKEL (19), National Insti- Johns Hopkins University, School of tute of Environmental Health Sciences, Medicine, Baltimore, Maryland 50212 National Institute of Health, Research Triangle Park, North Carolina 27709 JOHN D. NOTI (12), Molecular and Cell Biol- ogy, Triton Biosciences, Inc., Alameda, F. LEE (1), Department of Molecular Biol- California 10549 ogy, DNAX Research Institute of Molec- Mar and Cellular Biology, Palo Alto, Cali- H. OKAYAMA (1),' Laboratory of Cell fornia 94304 Biology, National Institute of Mental Health, Bethesda, Maryland 29802 COREY LEVENSON (21), Department of Chemistry, Cetus Corporation, Emery- DRAHCIR PINE (22), Department of Molecu- ville, California 94608 lar and BiCoellolg y, The Rockefeller Uni- versity, New York, New York 12001 DAVID F. (21), MARK Department of Molec- Mar Biology, Cetus Corporation, Emery- RODNAS ROGNOP (24), Institute of Enzymol- ville, California 94608 ogy, Hungarian Academy of Sciences, pf 7 Budapest 1502, Hungary, and Boyce M. MATTEUCCl (15), Genentech, Inc., Thompson Institute, Cornell University, South San Francisco, California 21149 Ithaca, New York 35841 HANS W. DJURHUUS MATTHES (14), La- boratoire de Gdndtique Moldculaire, AHTIHNARP (8), REDDY Department of Biol- ogy, Massachusetts Institute of Technol- LGME/CNRS et U.184/INSERM, Insti- ogy, Cambridge, Massachusetts 02139 tute de Chimie Biologique, Facult~ de M~decine, 67085 Strasbourg Cedex, A. A. (5), REYES Department of Molecular France Genetics, Beckman Research Institute of CONTRIBUTORS TO VOLUME 154 Xi the City of Hope, Duarte, California TRUEHEART JOSHUA (10), Whitehead Insti- 01019 tute for Biomedical Research, Cam- JOHN D. STREBOR (19), National Institute of bridge, Massachusetts 02142, and Mas- Environmental Health Sciences, National sachusetts Institute of Technology, Cam- Institute of Health, Research Triangle bridge, Massachusetts 93120 Park, North Carolina 90772 R. BRUCE WALLACE (5, 6), Department of MICHAEL HSABSOR (8), Department of Biol- Molecular Genetics, Beckman Research ogy, Brandeis University, Waltham, Mas- Institute of the City of Hope, Duarte, Cal- sachusetts 45220 ifornia 01019 KONRAD SCHWELLNUS (13), GBF (Gesell- ALICE WANG (21), Department of Molecu- schaft fiir Biotechnologische Forschung lar Biology, Cetus Corporation, Emery- mbH), Mascheroder Weg ,1 D-3300 ville, California 80649 Braunschweig, Federal Republic of Ger- EGROEG M. KCOTSNIEW (9), Department of many Biochemistry and Molecular Biology, The MICHAEL SMITH (17), Department of -DiB University of Texas Medical School at chemistry, University of British Colum- Houston, Houston, Texas 52277 bia, Vancover, British Columbia T. YOKOTA (1), Department of Molecular MICHAEL SNYDER (7), Department of -DiB Biology, DNAX Research Institute of Mo- chemistry, Stanford University School of lecular and Cellular Biology, Paid Alto, Medicine, Stanford, California 50349 California 40349 Z. YKSNIBATS (15), Department of Chemis- DRAHCIR A. YOUNG (7), Whitehead Insti- try and Biochemistry, University of Colo- tute for Biomedical Research, Cam- rado, Boulder, Colorado 90308 bridge, Massachusetts 02142, and De- ADRIEN STAUB (14), Laboratoire de -it~ndG partment of Biology, Massachusetts que Mol~culaire, LGME/CNRS et U.184/ Institute of Technology, Cambridge, INSERM, Institute de Biologique, Chimie Massachusetts 93120 Facultd de M~decine, 67085 Strasbourg Cedex, France DRAHCIR A. ZAKOUR (19), Molecular and Applied Genetics Laboratory, Allied Cor- SALGUOD SWEETSER (7), Whitehead Insti- poration, Morristown, New Jersey 06970 tute for Biomedical Research, Cam- bridge, Massachusetts 24120 MARK J. ZOLLER (17), Cold Spring Harbor Laboratory, Cold Spring Harbor. New ALADAR A. SZALAY (12), Boyce Thompson York 42711 Institute for Plant Research, Cornell Uni- versity, Ithaca, New York 35841 FRANS J. DE BRUIJN (ll), Max-Planck-lnsti- J.-Y. TAN(G1 5), Shanghai Institute of -DiB tut fiir Zi~chtungsforschung, Abteilung chemistry, Shanghai, Peoples Republic of Schell, D-5000 nlOK ,03 Federal Republic China of Germany JOHN W. TAYLOR (25), Laboratory of W.F. NAV GUNST (23), Department of Bioorganic Chemistry and Biochemistry, Physical Chemistry, University of Gro- The Rockefeller University, New York, ningen, 9747 AG Groningen, ehT Nether- New York 12001 lands [1] cDNA 3 CONSTRUCTION AND SCREENING OF LIBRARIES [1] High-Efficiency Cloning of Full-Length cDNA; Construction and Screening of cDNA Expression Libraries for Mammalian Cells By H. ,AMAYAKO M. ,IHCIAWAK M. ,NIETSNWORB F. ,EEL 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. 6-3 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, 59 (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. 451 All rights of reproduction in any form reserved. 4 SDOHTEM ROF GNINOLC 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 thep cD 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. 41-8 In fact, Yokota et al. 21,11 and Lee et al. 41,31 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 71 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). 01 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). 21 T. Yokota, N. Arai, F. Lee, D. Rennick, T. Mosmann, and K. Arai, Proc. Natl. Acad. Sci. U.S.A. 82, 68 (1985). 31 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). 41 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). 51 G. L. McKnight and B. C. McConaughy, Proc. Natl. Acad. Sci. U.S.A. 80, 4412 (1983). 61 A. Miyajima, N. Nakayama, I. Miyajima, N. Arai, H. Okayama, and K. Arai, Nucleic Acids Res. 12, 6639 (1984). 71 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 81 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 cloneo n the basis of the function it encodes, the library is acutelyt ransfectedo r 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 81 satisfies the above 81 j. M. Chiigwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, yrtsimehcoiB 18, 5294 (1978). 6 METHODS FOR CLONING cDNA [1] IN.O( 1519,01 im junctioe •ell Hiadlll -7"/ ~ C/at ~ Xhol ,. §o7.0,1 .v, "'°2'2(' Hindlll (0.71) FBR322 ori pBR~2 orl - /f o I ~.~ llldniH laS I i noO4VS / "~ ,,~'~ x~l a#l Awll ltsP pcO-X X(©DNA) 8P ~ ,~ 4 ~ t" ~1 & IHmm8 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 61 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 DNA GNINEERCS FO cDNA SEIRARBIL 7 ] VECTOR PRIMER I I OLIGO (dO) TAILED LINKER DNA ] )59.0( 1J]dniH )68.0( ItsP S61 ecilps ~ noitcnuj . Eco IR )857.0( pmA R // (0.93)~ X'h^' ~ )57.0{ AmpR(/// IVDcp "~ )457.0, f/ PstI-~ ]Lp ~ 04VS iro ~ InpK--~.)17.0( )517.0( \\ IHmaB (0.14~!~ XhoI 8 ~ ~ "polylA) ~~~./ dniH" ]]l )17.0( l91.0(" ~ PstI NOITSEGID KpnI NOITSEGID OGILO )Gd( SLIAT YLOP )Td( SLIAT /...-~ G G G Igd• niH ocE IR ~ ~ npK I ~ ~ /\ dniH IH Eco IR NOITSEGID I]ldniH NOITSEGID NOITACIFIRUP NOITACIFIRUP dniH ]]] ~ T OCE ohX I IR TTTT dniH nI Pstl G Xho I ~ Bam HI ~a .GIF .2 Preparation of linker and vector primer .sAND criteria and is reverse transcribed efficiently. It has successfully been used for cloning a number of cDNAs? 41- 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] ANRm . . . . ~ :GNILAENNA ~. Cc c .~ ANDc ~ - OGILO ,- % ~ SYNT.ES,S 'T~ SL,AT A ,,c _ [ f T ~ ~/ A.TT - ~ffll k T Hindlll Hindlll~Xh°l ~ lHmaB ~ REMIRP AC C C ,sP I f2 t "~ -- "~-AA /H~m H/ndlll ~GG G ¢/~ ,...~- -T T A J NOITSEGID mdniH lHmaB8 [ohX GNILAENNA OGIL OFO Gd ' DELIAT ;REKNIL NOITAZILCYC HTIW AND//oc.E ESAGIL / ~.G~..~ __~- ~ TNEMECALPER AN RFO STRAND ~ GGc~'~-- AND ESAREMYLOP AND 1 "~" A tl~ "~~: ~ ~ AND ESAGU ]Hd~'kH --'P~ AAT H/ndln .GIF .3 citamyznE steps ni eht noitcurtsnoc of pcD-cDNA .stnanibmocer -angised ehT snoit of eht AND stnemges era sa described ni Fig. .1 For latnemirepxe sliated dna -moc ,stnem see .sdohteM stnegaeR 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).