Recombinant DNA Part I

805 Pages·1993·13.811 MB·English

by  Abelson J. (ed.), Simon M. (ed.), Wu R. (ed.)

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).