Recombinant DNA Part B PDF
Preview Recombinant DNA Part B
Preface Exciting new developments in recombinant DNA research allow the isolation and amplification of specific genes or DNA segments from al- most any living organism. These new developments have revolutionized our approaches to solving complex biological problems and have opened up new possibilities for producing new and better products in the areas of health, agriculture, and industry. Volumes 100 and 101 supplement Volumes 65 and 68 of Methods in Enzymology. During the last three years, many new or improved methods on recombinant DNA or nucleic acids have appeared, and they are in- cluded in these two volumes. Volume 001 covers the use of enzymes in recombinant DNA research, enzymes affecting the gross morphology of DNA, proteins with specialized functions acting at specific loci, new methods for DNA isolation, hybridization, and cloning, analytical meth- ods for gene products, and mutagenesis: in vitro and in vivo. Volume 101 includes sections on new vectors for cloning genes, cloning of genes into yeast cells, and systems for monitoring cloned gene expression. RAY Wu LAWRENCE NAMSSORG KIVIE MOLDAVE iiix DRAHREG TDIMHCS 1901-1981 Gerhard Schmidt (1901-1981) This hundredth volume of Methods in Enzymology is dedicated to the memory of a dear friend and colleague whose pioneering work on the nucleic acids was important to the development of the techniques de- scribed in this and related volumes. Gerhard Schmidt was among the first to recognize the power of a combined chemical and enzymatic approach to the analysis of the structure of the nucleic acids. The importance of his work was belatedly recognized by his election to the National Academy of Sciences in 1976. In his classic work in 1928, while in Frankfurt in Embden's laboratory, he demonstrated the deamination of "muscle adenylic acid" by a highly specific enzyme which fails to deaminate "yeast adenylic acid." He speculated (correctly) that the two adenylic acids differed in the position of the phosphate group. He is probably best known for his development in 1945, while at the Boston Dispensary, of the method for determining the RNA, DNA, and phosphoproteins in tis- sues by phosphorus analysis (the Schmidt-Thannhauser method). He made many other contributions in the nucleic acid field, beginning with his studies with P. A. Levene at the Rockefeller Institute in 1938-1939 on the enzymatic depolymerization of RNA and DNA, and extending into the 1970s when he published some of the first definitive work on the nature of DNA-histone complexes. Schmidt's research was by no means limited to the nucleic acids. He was almost equally involved in studies on the structure and measurement of the complex lipids. He also made important observations on the accu- mulation of inorganic polyphosphates in living cells. During the period between his forced flight from Germany in 1933 when the Nazis came to power and his employment by Thannhauser at the Boston Dispensary in 1940, he had a variety of research fellowships in Italy, Sweden, Canada, and the United States, including one in 1939-1940 in the laboratory of Cad and Gerty Cori in St. Louis, where he worked on the enzymatic breakdown of glycogen by the muscle and liver phosphorylases. It was during this St. Louis period that one of us (SPC), then a gradu- ate student in the Cori laboratory, came to know Gerhard intimately. In the mid-1940s, the other one of us (NOK), then a postdoctoral fellow with Fritz Lipmann at the Massachusetts General Hospital, also developed close scientific and personal ties with Gerhard. In the early 1950s, when we had joined the McCollum-Pratt Institute, Gerhard was invited to par- ticipate in the Symposia on Phosphorus Metabolism where he presented a XXV xxvi DRAHREG TDIMHCS monumental review on the polyphosphates and metaphosphates, and was also a central figure in the discussions on the nucleic acids. In the late s0591 and the 1960s, when NOK returned to Boston to be on the Brandeis faculty, the close ties with Gerhard were renewed. In the early 1960s, shortly after SPC joined the Vanderbilt faculty, Gerhard was invited there as a visiting professor and gave a series of memorable lectures on the nucleic acids which also formed the basis for his typically thorough chap- ter on that subject which appeared in Annual Reviews of Biochemistry for .4691 During all the years from 1940 on, Gerhard did his research at the Boston Dispensary where Thannhauser had established a clinical chemis- try laboratory. Throughout that time, Gerhard also held a joint appoint- ment in biochemistry at the Tufts University School of Medicine where he participated in the teaching of medical students and the training of gradu- ate students. He enjoyed a good relationship with the successive Chair- men of that department, three of whom, Alton Meister, Morris Friedkin, and Henry Mautner, were especially helpful. Dr. Mautner was instrumen- tal in establishing the Gerhard Schmidt Memorial Lectureship which was initiated in December, 1981. Gerhard was one of the most universally beloved figures in biochemis- try. Perhaps this was because he lacked the "operator" gene. He would never have been comfortable as Chairman of a department or as President of a genetic engineering company. He liked to laugh, especiallayt himself. He identified with Laurel and Hardy, and once injured his jaw while rocking with laughter at one of their movies. He had a delightful collection of anecdotes, which, like his lectures, were carefully constructed and overly lengthy, but always well received by the Schmidt-story afficiona- dos. He was enthusiastic about many things in addition to science, but he attacked with special gusto the playing of good chamber music or the eating of a good Liederkranz. We present this dedication to his wife, Edith, and his sons, Michael and Milton, all of whom he loved very much, perhaps even more than his science, his music, and his Liederkranz. YENDIS P. COLOWICK NATHAN O. KAPLAN Contributors to Volume 100 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. A. REKCEB (12), Department of Medical -eG Medicine, University of Washington, netics, University of Toronto, Toronto, Seattle, Washington 59189 Ontario M5S 1A8, Canada RETEP T. (19), CHERaAS Department of -lleC LEAHCIM D. BEEN (8), Department of Mi- and ular Developmental Biology, Bi- ehT crobiology and Immunology, School of ological Laboratories, Harvard Univer- Medicine, University of Washington, sity, Cambridge, Massachusetts 83120 Seattle, Washington 59189 YNAOJ CHOU (21), Department of Biophys- DLAREG A. (19), BELTZ Department of -lleC ics and Biology, Theoretical University of ular and Developmental Biology, e-hiTB Chicago, Chicago, Illinois 73606 ological Laboratories, Harvard Univer- R. JOHN COLLIER (25), Department of Mi- sity, Cambridge, Massachusetts 83120 and crobiology ehT Biology In- Molecular H. C. BIRNBOIM (17), Radiation Biology stitute, University of California, Los Branch, Atomic Energy of Canada Lim- Angeles, California 42009 ited, Chalk River, Ontario KOJ IJO, SALOHCIN R. !LLERAZZOC (11), Department adanaC of Molecular Biology, University of -ilaC TREBOR YELSEKALB (1, 26), Bethesda Re- fornia, Berkeley, California 02749 search Laboratories, Inc., Gaithersburg, TREBLA E. GREBLHAD (23), Division of -loiB Maryland 77802 ogy and Medicine, Brown University, DAVID NIETSTOB (31), Department of Biol- Providence, Rhode Island 21920 ogy, Massachusetts Institute of Technol- NER-OUG DENG (5), Section of Biochemis- ogy, Cambridge, Massachusetts 93120 try, Molecular and Cell Biology, Cornell ENIREHTAC A. BRENNAN (2), Department University, Ithaca, New 3584York 1 of Biochemistry, School of Basic Medical ALAN DNOMAID (30), Sidney Farber Cancer Sciences and School of Chemical Sci- Institute and Harvard Medical School, ences, University of Illinois, Urbana, Illi- Boston, Massachusetts 51120 nois 10816 JOHN E. NOSLENOD (6), Department of -DiB ATINOB J. BREWER (8), Department of -eG chemistry, University of Iowa, Iowa City, netics, University of Washington, Seattle, Iowa 24225 Washington 59189 DAVID R. BROWN (16), Department of De- K. DORAN (26), Bethesda Research Lab- velopmental Biology and Cancer, Albert oratories, Inc., Gaithersburg, Maryland Einstein College of Medicine, Bronx, New 77802 York 16401 DRANREB KCODUD (30), Department of -DiB SNAH NNAMENOB (27), Institutfiir Genetik, chemistry, State University of New York, ti~tisrevinU D~isseldorf, D-4000 Diissel- Stony Brook, New York 49711 doff, Federal Republic of Germany SAMOHT H. HSUBKCIE (19), Department of MLOCLAM J. NABADASAC (21), Department Biology, University of Rochester, Roch- of Biophysics and Theoretical Biology, ester, New York 72641 University of Chicago, Chicago, Illinois TRAUTS G. (29), FISCHER Department of Bi- 73606 ological Sciences, Center for Biological SEMAJ J. XUOPMAHC (8), Department of Mi- Macromolecules, State University of New crobiology and Immunology, School of ,kroY Albany, New 2222York 1 ix X CONTRIBUTORS TO VOLUME 100 EmCH FREI (22), Department of Cell Biol- TAO-SHIH HSIEH (10), Department of Bio- ogy, Biocenter of the University, 6504-HC chemistry, Duke University Medical Cen- Basel, Switzerland ter, Durham, North Carolina 01772 RoY Fucns (1), Corporate Research and JERARD HURWITZ (16), Department of De- Development, Monsanto Company, St. velopmental Biology and Cancer, Albert Louis, Missouri 66136 Einstein College of Medicine, Bronx, New York 16401 JAMES I. GARRELS (28), Spring Cold Harbor Laboratory, Cold Spring Harbor, New KENNETH A. JACOBS (19), Department of York 42711 and Developmental Cellular Biology, The Laboratories, Biological Harvard Univer- M. GOLD (12), Department of Medical Ge- sity, Cambridge, Massachusetts 83120 netics, University of Toronto, Toronto, Ontario M5S 1A8, Canada CORNELIS VICTOR JONGENEEL (9), Depart- ment of Biochemistry~Biophysics, Univer- PETER GOWLAND (22), Department of Cell sity of California, San Francisco, San Biology, Biocenter of the University, Francisco, California 34149 6504-HC Basel, Switzerland FOTIS C. KAFATOS (19), Department of -lleC LAWRENCE GREENFIELD (25), Cetus Corpo- ular and Developmental Biology, ehT Bi- ration, Berkeley, California 01749 ological Laboratories, Harvard Univer- MANUEL GREZ (20), Department of Micro- sity, Cambridge, Massachusetts 83120 biology, University of Southern Califor- DONALD A. KAPLAN (25), Cetus Corpora- ain School of Medicine, Los Angeles, tion, Berkeley, California 01749 California 33009 KENNETH N. KREUZER (9), Department of RICHARD I. GUMPORT (2), Department of Biochemistry/Biophysics, University of Biochemistry, School of Basic Medical California, San Francisco, San Fran- Sciences and School of Chemical Sci- cisco, California 34149 ences, University of Illinois, Urbana, Illi- nois 10816 JuDY H. KRUEGER (33), Department of Bi- ology, Massachusetts Institute of Tech- LI-HE Guo (4), Section of Biochemistry, nology, Cambridge, Massachusetts 93120 Molecular and Biology, Cell Cornell Uni- versity, Ithaca, New York 35841 HARTMUT LAND (20), Center of Cancer Re- search, Massachusetts Institute of Tech- DOUGLAS HANAHAN (24), Department of nology, Cambridge, Massachusetts 93120 Biochemistry and Molecular Biology, Harvard University, Cambridge, Massa- ABRAHAM LEVY (22), Friedrich-Meischer- chusetts 02138, and Cold Spring Harbor Institut, Ciba-Geigy, CH-4058 Basel, Laboratory, Cold Spring Harbor, New Switzerland York 42711 WERNER LINDENMAIER (20), Gesellschaft JAMES L. HARTLEY (6), Bethesda Research fiir Biotechnologische Forschung, Ma- Laboratories Inc., Gaithersburg, Mary- scheroder Weg ,1 Braunschweig, D-3300 land 77802 Federal Republic of Germany HANSJt)RG HAUSER (20), Gesellschaft fiir LEROY F. LIU (7), Department of Physio- Biotechnologische Forschung, Maschero- Chemistry, Jological hns Hopkins Univer- der Weg ,1 D-3300 Braunschweig, Fed- sity Medical School, Baltimore, Maryland eral Republic of Germany 50212 C. J. HOUGH (26), Bethesda Research Labo- ALICE E. MANTHEY (2), Department of Bio- ratories, Inc., Gaithersburg, Maryland chemistry, School of Basic Medical Sci- 77802 ences and School of Chemical Sciences, CONTRIBUTORS TO VOLUME 100 xi University of Illinois, Urbana, Illinois Einstein College of Medicine, Bronx, New 10816 1640York 1 SUSAN R. MARTIN (8), Genetic Systems PAUL J. KUINAMOR (3), Department of Bio- First Corp., 3005 Avenue, Seattle, Wash- chemistry, University of Illinois, Urbana, ington 12189 Illinois 10816 OSNOFLA SAIRA-ZEN1TRAM (21), Depart- SAMOHT LEKNIWENEEG-TDIMHCS (16), De- ment of Biophysics and Theoretical Biol- partment of Developmental Biology and ogy, University of Chicago, Chicago, Illi- Cancer, Albert Einstein College of Medi- nois 73606 cine, Bronx, New York 16401 BETTY L. McCONAUGHY (8), Department REHTNOG SCHUTZ (20), Institut fiir Zell- of Genetics, University of Washington, und Tumorbiologie, Deutsches Krebsfor- Seattle, Washington, 59189 schungszentrum, lm Neuenheimer Feld WILLIAM K. McCoUBREY, JR. (8), Depart- ,082 0096-D Heidelberg, Federal "cilbupeR ment of Microbiology and Immunology, of Germany School of Medicine, University of Wash- STUART K. SHAPIRA (21), Committee on ington, Seattle, Washington 59189 Genetics, University of Chicago, Chi- MATTHEW NOSEESEM (24), Department of cago, Illinois 73606 Biochemistry and Molecular Biology, OKIHEKAT ATABIHS (14), Department of Mi- Harvard University, Cambridge, Massa- crobiology, ehT Institute of Physical and chusetts 83120 Chemical Research, Saitama 351, Japan DRAWOH A. (15), NASH Laboratory of Neu- DIVAD EETROHS (31), Department of Micro- rochemistry, National Institute of Mental biology, State University of New York, Health, Bethesda, Maryland 50202 Stony Brook, New York 49711 SUKRAM NOEL (22), Department of Bi- Cell LEAHCIM SMITH (32), Department of Bio- ology, Biocenter of the University, chemistry, Faculty of Medicine, Univer- 6504-HC Basel, Switzerland sity of Columbia, British Vancouver, Brit- ish Columbia V6T 1 ,SW Canada LYNN OSBER (14), Departments of Human Genetics, Yale School University of Medi- DNUMDE J. GAWLEETS (23), Department of cine, New Haven, Connecticut 01560 Microbiology, University of Minnesota, Minneapolis, Minnesota 55455 DRAHCIR OTTER (11), Department of Mo- Biology, lecular University of California, AICIRTAP S. SAMOHT (18), Genetic Systems Berkeley, California 02749 First Corporation, 3005 Avenue, Seattle, Washington 12189 W. SIRRAP (12), Department of Medical -eG netics, University of Toronto, Toronto, J. A. NOSPMOHT (26), Bethesda Research Ontario M5S 1A8, Canada Laboratories, Inc., Gaithersburg, Mary- land 77802 SELRAHC M. (14), RADDING Departments of Human Genetics and of Molecular Bio- OEKE C. UHLENBECK (3), Department of physics and Biochemistry, Yale Univer- Biochemistry, University of Illinois, -rU sity School of Medicine, New Haven, bana, Illinois 10816 Connecticut 01560 MAHARG C. WALKER (33), Department of LLADNAR R. REED (13), Department of -eG Biology, Massachusetts Institute of Tech- netics, Harvard Medical School, Boston, nology, Cambridge, Massachusetts 93120 Massachusetts 51120 TREBOR D. WELLS (26), Department of -DiB YNNAD GREBNIER (16), Department of De- chemistry, Schools of Medicine and Den- velopmental Biology and Cancer, Albert tistry, University of Alabama, Birming- xii CONTRIBUTORS TO VOLUME 100 ham, University Station, Birmingham, Biology, University of Oregon, Eugene, Alabama 35294 Oregon 30479 WESTHOFF PETER (27), Botanik IV, Univer- NEHPETS L. YKSRUPIZ (16), Division of Bi- Diisseldorf, sit?it Diisseldorf, D-4000 Fed- ology, California Institute of Technology, eral Republic of Germany Pasadena, California 52009 YAR Wu (4, 5), Section of Biochemistry, MARK J. ZOLLER (32), Department of Bio- Molecular and Cell Biology, Cornell Uni- chemistry, Faculty of Medicine, Univer- versity, Ithaca, New York 35841 sity of British Columbia, Vancouver, Brit- LISA S. YOUNG (8), Institute of Molecular ish Columbia V6T IW5, Canada 3 [1] USE OF TYPE II RESTRICTION ENDONUCLEASES [1] Guide to the Use of Type II Restriction Endonucleases By RoY FUCHS and ROBERT YELSEKALB Type II restriction endonucleases are DNases that recognize specific oligonucleotide sequences, make double-strand cleavages, and generate unique, equal molar fragments of a DNA molecule. By the nature of their controllable, predictable, infrequent, and site-specific cleavage of DNA, restriction endonucleases proved to be extremely useful as tools in dis- secting, analyzing, and reconfiguring genetic information at the molecular level. Over 350 different restriction endonucleases have been isolated from a wide variety of prokaryotic sources, representing at least 85 differ- ent recognition sequences.~.2 A number of excellent reviews detail the variety of restriction enzymes and their sources, 3,2 their purification and determination of their sequence specificity, 5,4 and their physical proper- ties, kinetics, and reaction mechanism. 6 Here we provide a summary, based on the literature and our experience in this laboratory, emphasizing the practical aspects for using restriction endonucleases as tools. This review focuses on the reaction, its components and the conditions that affect enzymic activity and sequence fidelity, methods for terminating the reaction, some reaction variations, and a troubleshooting guide to help identify and solve restriction endonuclease-related problems. The Reaction Despite the diversity of the source and specificity for the over 350 type II restriction endonucleases identified to date, 2L their reaction conditions are remarkably similar. Compared to other classes ofe nzymes these con- ditions are also very simple. The restriction endonuclease reaction (Ta- ble I) is typically composed of the substrate DNA incubated at ° 37 in a solution buffered near pH 7.5, containing Mg ,÷2 frequently Na ÷, and the selected restriction enzyme. Specific reaction details as found in the liter- I R. Blakesley, in "Gene Amplification and Analysis," Vol. :1 "Restriction Endonu- cleases" (J. G. Chirikjian, ed.), p. .1 Elsevier/North-Holland, Amsterdam, 1981. 2 R. J. Roberts, Nucleic Acids Res. 10, rl17 (1982). 3 j. G. Chirikjian, "Gene Amplification and Analysis," Vol. :1 "Restriction Endonu- cleases." Elsevier/North-Holland, Amsterdam, 1981. 4 R. J. Roberts, CRC Crit. Reo. Biochem. 4, 321 (1976). 5 This series, Vol. 65, several articles. 6 R. D. Wells, R. D. Klein, and C. K. Singleton, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 14, Part A, p. 157. Academic Press, New York, 1981. Copyright © 1983 by Academic Press, inc. METHODS IN ENZYMOLOGY, VOL. 001 All rights of reproduction in any form reserved. ISBN 0-12-182000-9 4 SEMYZNE NI TNANIBMOCER DNA ]1[ TABLE I DEZILARENEG REACTION SNOITIDNOC ROF NOITCIRTSER SESAELCUNODNE Reaction type Conditions Analytical Preparative Volume 20-100/xl 0.5-5 ml DNA 0.1-10/zg 10-500/zg Enzyme 1-5 units//zg DNA 1-5 units//zg DNA Tris-HCl (pH 7.5) 20-50 mM 50 mM MgCI2 5-10 mM 01 mM 2-Mercaptoethanol 5-10 mM 5-10 mM Bovine serum albumin 50-500/zg/ml 200-500/~g/ml Glycerol <5% (v/v) <5% (v/v) NaCI As required As required Time 1 hr 1-5 hr Temperature ° 37 ° 37 ature for the more frequently used enzymes are listed in Table II. Note that in most cases these data do not represent optimal reaction conditions. By convention, a unit of restriction endonuclease activity is usually defined as that amount of enzyme required to digest completely 1 g~/ of DNA (usually of bacteriophage lambda) in 1 hr. 4 This definition was cho- sen for convenience, since the useful, readily measurable end result of a restriction endonuclease reaction is completely cleaved DNA. However, a unit defined in this manner measures enzyme activity by an end point rather than by the classical initial rate term. Thus, traditional kinetic arguments based upon substrate saturating (initial rate) conditions cannot be applied to restriction endonucleases defined in this (enzyme saturating) manner. One reason why there are few proper kinetic data on restriction en- donucleases lies in the difficulty in measuring restriction enzyme activi- ties during the linear portion of the reaction when using the standard enzyme assay. 7 The strong emphasis placed on their use as research tools in molecular biology rather than on investigation of their biochemical properties also contributed to the deficiency. Hence we lack good experi- mental data on conditions for optimal activity. For most newly isolated restriction endonucleases, assay buffers were selected for convenience during enzyme isolation rather than for optimal reactivity. These condi- tions have persisted as dogma. Thus, the implied precision and unique- 7 p. A. Sharp, B. Sugden, and J. Sambrook, Biochemistry 12, 3055 (1973).
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