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 from any source or their regulatory regions can be modified at will and reintroduced into a wide variety of cells by transformation. The cells expressing the introduced gene can be measured at the RNA level or protein level. These advantages allow us to solve complex biolog- ical problems, including medical and genetic problems, and to gain deeper understandings at the molecular level. In addition, new recombinant DNA methods are essential tools in the production of novel or better products in the areas of health, agriculture, and industry. The new Volumes 216, 217, and 812 supplement Volumes 153, 154, and 551 of Methods ni Enzymology. During the past few years, many new or improved recombinant DNA methods have appeared, and a number of them are included in these new volumes. Volume 612 covers methods related to isolation and detection of DNA and RNA, enzymes for manipu- lating DNA, reporter genes, and new vectors for cloning genes. Volume 712 includes vectors for expressing cloned genes, mutagenesis, identify- ing and mapping genes, and methods for transforming animal and plant cells. Volume 812 includes methods for sequencing DNA, PCR for ampli- fying and manipulating DNA, methods for detecting DNA-protein inter- actions, and other useful methods. Areas or specific topics covered extensively in the following recent volumes of Methods ni Enzymology are not included in these three vol- umes: "Guide to Protein Purification," Volume 182, edited by M. P. Deutscher; "Gene Expression Technology," Volume 185, edited by D. V. Goeddel; and "Guide to Yeast Genetics and Molecular Biology," Volume 194, edited by C. Guthrie and G. R. Fink. YAR UW iivx Contributors to Volume 218 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. MARIE ALLEN (1), Department of Medical IGOR BRIKUN (19), Department of Molecu- Genetics, University of Uppsala Biomedi- ral Microbiology, Washington University cal Center, S-751 32 Uppsala, Sweden School of Medicine, St. Louis, Missouri OCSICNARF Josf~ AYALA (21), Department 01136 of Organismic and Evolutionary Biology, CAROL M. BROWN (13), Medical Research Harvard University, Cambridge, Massa- Council Laboratory of Molecular Biol- chusetts 83120 ogy, Cambridge 2BC 2QH, England ALAN T. BANKIER (13), Medical Research MICHAEL BULL (22), Department of Immu- Council Laboratory of Molecular Biol- nology, Mayo Clinic, Rochester, Minne- ogy, Cambridge 2BC 2QH, England sota 50955 GLADYS I. CASSAB (48), Plant Molecular Bi- BARCLAY G. BARRELL (13), Medical Re- ology and Biotechnology, Institute of Bio- search Council Laboratory of Molecular technology, National Autonomous Uni- Biology, Cambridge 2BC 2QH, England versity of Mexico, Cuernavaca ,17226 STEVEN R. BAUER (33), Laboratory of Mo- Mexico lecular Immunology, Department of JOSLYN D. CASSADY (29), Department of Health and Human Services, Food and Biochemistry and Molecular Biology, Drug Administration, Bethesda, Mary- Mayo Clinic~Foundation, Rochester, land 29802 Minnesota 50955 PETER B. BECKER (40), Gene Expression MARK S. CHEE (13), Affymax Research In- Program, European Molecular Biology stitute, Palo Alto, California 40349 Laboratory, D-6900 Heidelberg, Ger- CATHIE T. CHUNG (43), Hepatitis Viruses many Section, Laboratory of Infectious Dis- MICHAEL BECKER-ANDRI~ (32), GLAXO In- eases, National Institute of Allergy and stitute for Molecular Biology, 1228 Plan- Infectious Diseases, National Institutes les-Ouates, Geneva, Switzerland of Health, Bethesda, Maryland 29802 CLAIRE M. BERG (19, 20), Departments of GEORGE M. CHURCH (14), Department of Molecular and Cell Biology, University of Genetics, Howard Hughes Medical Insti- Connecticut, Storrs, Connecticut 96260 tute, Harvard Medical School, Boston, DOUGLAS E. BERG (19, 20), Departments of Massachusetts 51120 Molecular Microbiology and Genetics, JOHN A. CIDLOWSKI (38), Department of Washington University School of Medi- Physiology, University of North Carolina cine, St. Louis, Missouri 01136 at Chapel Hill, Chapel Hill, North Caro- CYNTHIA D. K. BOTTEMA (29), Department lina 99572 of Biochemistry and Molecular Biology, MOLLY CRAXTON (13), Medical Research Mayo Clinic~Foundation, Rochester, Council Laboratory of Molecular Biol- Minnesota 50955 ogy, Cambridge 2BC 2QH, England SYDNEY BRENNER (18), Department of PETER B. DERVAN (15), Arnold and Mabel Medicine, Cambridge University, Cam- Beckman Laboratories of Chemical Syn- bridge 2BC 2QH, England, and ehT thesis, Division of Chemistry and Chemi- Scripps Research Institute, La Jolla, Cal- cal Engineering, California Institute of ifornia 12129 Technology, Pasadena, California 52119 xi xii CONTRIBUTORS TO VOLUME 218 CRAIG A. DIONNE (30), Cephalon, Inc., WALTER GILBERT (4), Department of -ulleC West Chester, Pennsylvania 08391 ral and Developmental Biology, Harvard DAVID M. DOREMAN (23), Department of University, Cambridge, Massachusetts Pathology, Brigham and Women's Hospi- 83120 tal, Boston, Massachusetts 02115, and JACK GORSKI (22), Immunogenetics Re- Harvard Medical School, Harvard Uni- search Section, Blood Research Institute, versity, Cambridge, Massachusetts 83120 The Blood Center of Southeastern Wis- consin, Milwaukee, Wisconsin 33235 ROBERT L. DORIT (4), Department of Biol- ogy, Yale University, New Haven, Con- MICHAEL M. GOTTESMAN (45), Laboratory necticut 11560 of Cell Biology, National Cancer Insti- tute, National Institutes of Health, Be- HOWARD DROSSMAN 02), Department of thesda, Maryland 29802 Chemistry, Colorado College, Colorado Springs, Colorado 30908 RICHARD W. GROSS (17), Division of Bioorganic Chemistry and Molecular ZIJIN Du (10), Department of Genetics, Pharmacology, Washington University Washington University School of Medi- School of Medicine, St. Louis, Missouri cine, St. Louis, Missouri 01136 01136 CHARYL M. DUTTON (29), Department of TOM J. GUILEOYLE (49), Department of -DiB Biochemistry and Molecular Biology, chemistry, University of Missouri, -oC Mayo Clinic~Foundation, Rochester, erul bia, Missouri 11256 Minnesota 50955 ULF B. GYLLENSTEN (1), Department of DAVID D. ECKELS (22), lmmunogenetics Medical Genetics, University of Uppsala Research Section, Blood Research Insti- Biomedical Center, S-751 32 Uppsala, tute, The Blood Center of Southeastern Sweden Wisconsin, Milwaukee, Wisconsin 33235 PERRY B. HACKETT (5), Department of -eG FRITZ ECKSTEIN (8), Abteilung Chemie, netics and Cell Biology, University of Max-Planck-Institut fiir Experimentelle Minnesota, St. Paul, Minnesota 80155 Medizin, D-3400 G6ttingen, Germany GRETCHEN HAGEN (49), Department of -DiB HENRY ERLICH (27), Department of Human chemistry, University of Missouri, -oC Genetics, Roche Molecular Systems, Ala- lumbia, Missouri 11256 meda, California 10549 MICHAEL K. HANAFEY (51), Agricultural JAMES A. FEE (50), Spectroscopy and Bio- Products Department, E. I. DuPont de chemistry Group, Isotope and Nuclear Nemours & Company, Wilmington, Dela- Chemistry Division, Los Alamos National ware 08891 Laboratory, Los Alamos, New Mexico DANIEL L. HARTL (3, 21), Department of 54578 Organismic and Evolutionary Biology, MICHAEL A. FROHMAN (24), Department of Harvard University, Cambridge, Massa- Pharmacological Sciences, State Univer- chusetts 83120 sity of New York at Stony Brook, Stony CHERYL HEINER (11), Applied Biosystems, Brook, New York 49711 Inc., Foster City, California 40449 ODD S. GABRIELSEN (36), Department of LEROY HOOD (10, 11), Department of Mo- Biochemistry, University of Oslo, N-0316 lecular Biotechnology, School of Medi- Oslo, Norway cine, University of Washington, Seattle, MELISSA A. GEE (49), Worcester Founda- Washington 59189 tion for Experimental Biology, Shrews- BRUCE H. HOWARD (45), Laboratory of Mo- bury, Massachusetts 54510 lecular Growth Regulation, National In- MARY JANE GEIGER (22), Department of stitute of Child Health and Human Devel- Medicine, Duke University Medical Cen- opment, National Institutes of Health, ter, Durham, North Carolina 01772 Bethesda, Maryland 29802 CONTRIBUTORS TO VOLUME 218 XII1 .°° TAZUKO HOWARD (45), Laboratory of Cell J. ANDREW KEIGHTLEY (50), Spectroscopy Biology, National Cancer Institute, Na- and Biochemistry Group, Isotope and Nu- tional Institutes of Health, Bethesda, clear Chemistry Division, Los Alamos Maryland 29802 National Laboratory, Los Alamos, New HENRY V. HUANG (19), Department of Mo- Mexico 54578 lecular Microbiology, Washington Uni- DAVID J. KEMP (37), Menzies School of versity School of Medicine, St. Louis, Health Research, Casuarina, Northern Missouri 01136 Territory 0811, Australiu JANINE HUET (36), Service de Biochimi et DANGERUTA KERSULYTE (19), Department G~n~tique Mol~culaire, Centre d'Etudes of Molecular Microbiology, Washington ed Saclay, 19119 Gif-sur-Yvette, France University School of Medicine, St. Louis, Missouri 01136 TIM HUNKAPILLER (11), Department of Mo- BRUCE C. KLINE (26), Department of Bio- lecular Biotechnology, School of Medi- chemistry and Molecular Biology, Mayo cine, University of Washington, Seattle, ,noitadnuoF~cinilC Rochester, Minnesota Washington 59189 50955 NORIO ICHIKAWA (46), Department of -DiB TONY KOSTICHKA (12), CAD', North Caro- chemistry, School of Hygiene and Public lina 11572 Health, The Johns Hopkins University, Baltimore, Maryland 50212 JAN P. KRAUS (16), Department of Pediat- rics, University of Colorado School of SETSUKO h (29), Department of Biochemis- Medicine, Denver, Colorado 26208 yrt and Molecular Biology, Mayo Clinic/ MARTIN KREITMAN (2), Department of Foundation, Rochester, Minnesota 50955 Ecology and Evolution, University of -ihC BRENT L. IVERSON (15), Arnold and Mabel cago, Chicago, Illinois 73606 Beckman Laboratories of Chemical Syn- KEITH A. KRETZ (7), Department of thesis, Division of Chemistry and Chemi- Neurosciences and Center for Molecular cal Engineering, California Institute of Genetics, School of Medicine, University Technology, Pasadena, California 52119 of California, San Diego, La Jolla, Cali- MICHAEL JAYE (30), Department of Molecu- fornia 39029 lar Biology, Rh6ne-Poulenc Rorer Cen- B. RAJENDRA KRISHNAN (19), Department tral Research, Collegeville, Pennsylvania of Medicine, Washington University 62491 School of Medicine, St. Louis, Missouri D. S. C. JONES (9), Medical Research Coun- 01136 cil, Molecular Genetics Unit, Cambridge LAURA F. LANDWEBER (2), Department of 2BC 2QH, England Cellular and Developmental Biology, Bio- logical Laboratories, Harvard University, MICHAEL D. JONES (31), Department -loriV Cambridge, Massachusetts 83120 ogy, Royal Postgraduate Medical School, Hammersmith Hospital, University of JEFFREY G. LAWRENCE (3), Department of London, London 21W ONN, England Biology, University of Utah, Salt Lake City, Utah 21148 VINCENT JUNG (25), Cold Spring Harbor Laboratories, Cold Spring Harbor, New JEAN-CLAUDE LELONG (42), lnstitut d'On- cologie Cellulaire et Moldculaire Hu- York 42711 maine, Universitd ed Paris Nord, 00039 ROBERT KAISER (11), Department of Molec- Paris, France ular Biotechnology, School of Medicine, ANDREW M. LEW (37), Burnet Clinical Re- University of Washington, Seattle, Wash- search Unit, The Walter and Eliza Hall ington 59189 Institute of Medical Research, Royal Mel- ERNEST KAWASAKI (27), Procept, Inc., bourne Hospital, Parkville, Victoria ,0503 Cambridge, Massachusetts 93120 Australia xiv CONTRIBUTORS TO VOLUME 218 ZHANJIANG LIU (5), Institute of Human Ge- DAVID B. OLSEN (8), Merck Sharp and netics, University of Minnesota, St. Paul, Dohme Research Laboratories, West Minnesota 55108 Point, Pennsylvania 68491 KENNETH J. LIVAK (18), DaPont Merck R. PADMANABHAN (45), Department of Bio- Pharmaceutical Company, Wilmington, chemistry and Molecular Biology, Uni- Delaware 08891 versity of Kansas Medical Center, Kan- sas City, Kansas 30166 MATTHEW J. LONGLEY (41), Department of Biochemistry, Duke University Medical RAJI PADMANABHAN (45), Department of Health and Haman Services, National In- Center, Durham, North Carolina 01772 stitutes of Health, Bethesda, Maryland JOHN A. LUCKEY (12), Department of 29802 Chemistry, University of Wisconsin- SIDNEY PESTKA (25), Department of Molec- Madison, Madison, Wisconsin 60735 raM Genetics & Microbiology, University V1KKI M. MARSHALL (37), Immunoparasi- of Medicine and Dentistry of New Jersey, tology Unit, The Walter and Eliza Hall Robert Wood Johnson Medical School, Institute of Medical Research, Royal Mel- Piscataway, New Jersey 45880 bourne Hospital, Parkville, Victoria ,0503 STEVEN B. PESTKA (25), North Caldwell, Australia New Jersey 60070 MICHAEL W. MATHER (50), Department of MICHAEL GREGORY PETERSON (35), Talarik, Biochemistry and Molecular Biology, Inc., South San Francisco, California Oklahoma State University, Stillwater, 08049 Oklahoma 87047 JAMES W. PRECUP (26), Department of Bio- BRUCE A. McCLURE (49), Department of chemistry and Molecular Biology, Mayo Biochemistry, University of Missouri, ,noitadnuoF~cinilC Rochester, Minnesota Columbia, Missouri 11256 50955 TERRi L. McGUIGAN (18), DuPont Merck J. ANTONI RAFALSKI (51), Agricultural Pharmaceutical Company, Wilmington, Products Department, E. .1 DuPont de Delaware 08891 Nemours & Company, Wilmington, Dela- ware 08891 ROGER H. MILLER (43), Hepatitis Viruses Section, National Institute of Allergy and WILLIAM D. RAWL1NSON (13), Medical Re- search Council Laboratory of Molecular Infectious Diseases, National Institutes Biology, Cambridge CB2 2QH, England of Health, Bethesda, Maryland 29802 PETER RICHTERICH (14), Department of Hu- DALE W. MOSBAUGH (41), Departments of man Genetics and Molecular Biology, Agricultural Chemistry, Biochemistry, evitaroball?~C Research, Inc., Waltham, and Biophysics, Oregon State University, Massachusetts 45120 Corvallis, Oregon 13379 RANDALL SAIKI (27), Department of Hu- JOHN S. O'BRIEN (7), Department of man Genetics, Roche Molecular Systems, Neurosciences and Center for Molecular Alameda, Calfornia 10549 Genetics, School of Medicine, University GURPREET S. SANDHU (26), Department of of California, San Diego, La Jolla, Cali- Biochemistry and Molecular Biology, fornia 39029 Mayo Clinic~Foundation, Rochester, HOWARD OCHMAN (3, 21), Department of Minnesota 50955 Biology, University of Rochester, GOBINDA SARKAR (28, 29), Department of Rochester, New York 72641 Biochemistry and Molecular Biology, OSAMU OHARA (4), Shinogi Research Labo- Mayo Clinic~Foundation, Rochester, ratories, Osaka, Japan Minnesota 50955 CONTRIBUTORS TO VOLUME 218 XV RICHARD H. SCHEUERMANN (33), Depart- SCOTT V. TINGLY (51), Agricultural Prod- ment of Pathology, University of Texas ucts Department, E. I. DuPont ed Ne- Southwestern Medical Center, Dallas, mours & Company, Wilmington, Dela- Texas 53257 ware 08891 J. P. SCHOEIELD (9), Medical Research ROBERT TJIAN (35), Howard Hughes Medi- Council, Molecular Genetics Unit, Cam- cal Institute, Department of Molecular bridge 2BC 2QH, England and Cell Biology, University of Califor- GONTHER SCHOTZ (40), Institute of Cell and nia, Berkeley, Berkeley, California 02749 Tumor Biology, German Cancer Re- PAUL O. P. Zs'o (46), Department of Bio- search Center, D-6900 Heidelberg, Ger- chemistry, School of Hygiene and Public many Health, The Johns Hopkins University, WENYAN SHEN (6), Whitehead Institute, Baltimore, Maryland 50212 Cambridge, Massachusetts 24120 DOUGLAS B. TULLY (38), Department of HARINDER SINGH (39), Department of Mo- Physiology, University of North Carolina lecular Genetics and Cell Biology, at Chapel Hill, Chapel Hill, North Caro- Howard Hughes Medical Institute, Uni- lina 99572 versity of Chicago, Chicago, Illinois 73606 ANGELA UY (8), Abteilung Medizinische Mikrobiologie des Zentrums fiir Hygiene LLOYD M. SMITH (12), Department of und Humangenetik der Universitiit, D- Chemistry, University of Wisconsin- 0043 GOttingen, Germany Madison, Madison, Wisconsin 60735 VICTORIA SMITH (13), Department of -eG JOSEPH E. VARNER (47), Department of Bi- ology, Washington University, St. Louis, netics, Stanford University, Stanford, California 50349 Missouri 03136 HANS SODERLUND (34), Biotechnical Labo- M. VAUDIN (9), Medical Research Council, ratory, Technical Research Centre of Fin- Molecular Genetics Unit, Cambridge 2BC land, 02150 Espoo, Finland 2QH, England STEVE S. SOMMER (28, 29), Department of GAN WANG (20), Department of Molecular Biochemistry and Molecular Biology, and Cell Biology, University of Connecti- Mayo Clinic~Foundation, Rochester, cut, Storrs, Connecticut 96260 Minnesota 50955 MARY M. Y. WAVE (6), Department of Bio- YAH-Ru SONG (47), Department of Plant chemistry, The Chinese University of Physiology, Institute of Botany, Aca- Hong Kong, Shatin, New Territories, demia Sinica, Beijing ,44001 China Hong Kong DAVID L. STEFFENS (17), Research andDe- FALK WEIH (40), Department of Molecular velopment, Li-Cor, Inc., Lincoln, Ne- Biology, Bristol-Myers Squibb Pharma- braska 40586 ceutical Research Co., Princeton, New LINDA D. STRAUSBAUGH (20), Department Jersey 34580 of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 96260 PAUL A. WHITTAKER (44), Clinical Bio- chemistry, University of Southampton, ANN-CHRISTINE SYVANEN (34), Depart- and South Laboratory and Pathology ment of Human Molecular Genetics, Na- Block, Southampton General Hospital, tional Public Health Institute, 00300 Hel- Southampton S09 4XY, England sinki, Finland OR1HAKAT TAHARA (16), Department of Pe- JOHN G. K. WILLIAMS (51), Data Manage- diatrics, National Okura Hospital, Tokyo ment Department, Pioneer Hi-Bred Inter- ,751 Japan national, Johnston, Iowa 13105 xvi CONTRIBUTORS TO VOLUME 218 RICHARD K. WILSON (|0), Department of ZHEN~-HuA YE (47), Department of Biol- Genetics, Washington University School ogy, Washington University, St. Louis, of Medicine, St. Louis, Missouri 01136 Missouri 03136 GERD WUNDERLICH (8), Abteilung Medi- MING YI (46), Department of Biochemistry, zinische Mikrobiologie des Zentrums fiir School of Hygiene and Public Health, Hygiene und Hamangenetik der Universi- The Johns Hopkins University, Balti- tilt, D-3400 Gfttingen, Germany more, Maryland 50212 1 GNICNEUQES FO in Vitro DEIFILPMA DNA 3 1 Sequencing of in Vitro Amplified DNA By ULF B. GYLLENSTEN and MARIE ALLEN Introduction The polymerase chain reaction (PCR) 2'1 method for in vitro amplifica- tion of specific DNA fragments has opened up a number of fields in molecular biology that were previously intangible because of lack of suffi- ciently sensitive analytical methods. The PCR is based on the use of two oligonucleotides to prime DNA polymerase-catalyzed synthesis from opposite strands across a region flanked by the priming sites of the two oligonucleotides. By repeated cycles of DNA denaturation, annealing of oligonucleotide primers, and primer extension an exponential increase in copy number of a discrete DNA fragment can be achieved. Many applications of PCR, including diagnosis of heritable disorders, screening for susceptibility to disease, and identification of bacterial and viral patho- gens, require determination of the nucleotide sequence of amplified DNA fragments. In this chapter we review alternate methods for the generation of sequencing templates from amplified DNA and sequencing by the method of Sanger. 3 Generation of Sequencing Template for Direct Sequencing Traditionally, templates for DNA sequencing have been generated by inserting the target DNA into bacterial or viral vectors for multiplication of the inserts in bacterial host cells. These cloning methods have been simplified, but are still subject to inherent problems associated with the maintenance and use of systems dependent on living cells, such as de novo mutations in vector and host cell genomes. By using PCR, templates for sequencing can be generated more efficiently than with cell-dependent methods either from genomic targets or from DNA inserts cloned into vectors. Amplification of cloned inserts of unknown sequence can be achieved using oligonucleotides that are priming inside, or close to, the polylinker of the cloning vector. 2 Sequencing the PCR products directly has two advantages over se- I K. B. Mullis and F. Faloona, this series, Vol. 155, p. 335. 2 R. K. Saiki, D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. Science 239, 487 (1988). 3 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1979). thgirypoC © 3991 yb cimedacA Press, .cnI SDOHTEM NI ,YGOLOMYZNE .LOV 812 llA sthgir of noitcudorper ni yna mrof .devreser 4 SDOHTEM ROF GNICNEUQES DNA 1 quencing of cloned PCR products. First, it is readily standardized because it is a simple enzymatic process that does not depend on the use of living cells. Second, only a single sequence needs to be determined for each sample (for each allele). By contrast, when PCR products are cloned, a consensus sequence based on several cloned PCR products must be determined for each sample, in order to distinguish mutations present in the original genomic sequence from random misincorporated nucleotides introduced by the Taq polymerase during PCR. Optimization of Polymerase Chain Reaction Conditions for Direct Sequencing The ease with which clear and reliable sequences can be obtained by direct sequencing depends on the ability of the PCR primers to amplify only the target sequence (usually called the specificity of the PCR), and the method used to obtain a template suitable for sequencing. The specificity of the PCR is to a large extent determined by the sequence of the oligonucleo- tides used to prime the reaction. For an individual pair of primers the specificity of the PCR can be optimized by changing the ramp conditions, the annealing temperature, and the 21CgM concentration in the PCR buffer. A titration, in 0.2 mM increments, of 21CgM concentrations from 0.1 to 3.0 mM in the final reaction is advised if the standard 5.1 mM concentration fails to produce the necessary specificity of the PCR. In cases in which optimization of PCR conditions fails to produce the desired priming specificity, either new oligonucleotides are required or the different PCR products can be separated by gel electrophoresis and reamplified individually for sequencing. When the PCR primers amplify several related sequences of the same length, for example, the same exon from several recently duplicated genes, or repetitive or conserved signal sequences, electrophoretic separation of the different products can be achieved either by the use of restriction enzymes that cut only certain templates and subsequent gel purification of the intact PCR products, or by the use of an electrophoretic system (denaturing gradient gel electrophoresis, temperature gradient gel electro- phoresis) for separation that will differentiate between the products based on their nucleotide sequence difference. 5,4 4 .R .M Myers, .V .C ,dlemehS dna .D .R Cox, ni "Genome Analysis--A lacitcarP -pA proach" .K( .E ,seivaD ed.), .p .59 LRI ,sserP ,drofxO .8891 5 .V .C ,dlemehS .D .R ,xoC .L .S ,namreL dna .R .M ,sreyM Proc. Natl. Acad. Sci. U.S.A. ,68 232 .)9891( 1 GNICNEUQES FO ni ortiV DEIFILPMA DNA 5 Double-Stranded DNA Templates Many of the problems associated with direct sequencing of PCR prod- ucts are not due to lack of specificity, but result from the ability of the two strands of the linear amplified product to reassociate rapidly after denaturation, thereby either blocking the primer-template complex from extending or preventing the sequencing oligonucleotide from annealing efficiently. 6 This problem is more severe for longer PCR products. To circumvent the strand reassociation of double-stranded DNA (dsDNA), a number of alternate methods have been developed. Precipitation of Denatured DNA Denature the template in 0.2 M NaOH for 5 rain at room temperature, transfer the tube to ice, neutralize the reaction by adding 0.4 vol of 5 M ammonium acetate (pH 7.5), and immediately precipitate the DNA with 4 vol of ethanol. Resuspend the DNA in sequencing buffer and primer at the desired annealing temperature. 7 Snap-Cooling of Template DNA Denature the template by heating (95 )° for 5 rain. Quickly freeze the tube by putting it in a dry ice-ethanol bath to slow down the reassociation of strands. Add sequencing primer either prior to or after denaturation and bring the reaction to the proper temperature. 8 Cycling of Polymerase Chain Reactions A third method for generating enough sequencing template is to cycle the sequencing reaction, using qaT polymerase as the enzyme for both amplification and sequencing. Even though only a small fraction of the templates will be utilized in each round of extension-termination, the amount of specific terminations will accumulate with the number of cycles. 01-8 6 U. B. Gyllensten, and H. A. Erlich, Proc. Natl. Acad. Sci. U.S.A. 85, 7652 (1988). v L. A. Wrischnik, R. G. Higuchi, M. Stoneking, H. A. Erlich, N. Arnhein, and A. C. Wilson, Nucleic Acids Res. 15, 529 (1987). 8 N. Kusukawa, T. Uemori, K. Asada, and I. Kato, Biotechniques 9, 66 (1990). 9 M. Craxton, Methods: Companion Methods Enzymol. 3, 20 (1991). 0l J.-S. Lee, DNA 10, 67 (1991).