Recombinant DNA Part G

677 Pages·1992·11.72 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 218 supplement Volumes 153, 154,
and 551 of Methods in 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 216 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
217 includes vectors for expressing cloned genes, mutagenesis, identify-
ing and mapping genes, and methods for transforming animal and plant
cells. Volume 218 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 in 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 Wu
XV
Contributors to Volume 216
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
ROBIN C. ALLSHIRE (51), MRC Human -eG KATHRYN J. BOCKHOLD (17), Ddpartment
netics Unit, Western General Hospital, ed Biologie Mol~culaire, Institut Pasteur,
Edinburgh EH4 2XU, Scotland 42757 Paris Cedex ,51 France
J. ALTENBUC~INER (40), Institute of Indus- JOHAN BOTTERMAN (36), Plant Genetics
trial Genetics, University of Stuttgart, Systems, B-9000 Gent, Belgium
0007-D Stuttgart ,1 Germany ALLAY R. BRASIER (34), Division of Endo-
MICHELLE A. ALTING-MEES (42), Strate- crinology and Hypertension, University
gene Cloning Systems, La Jolla, Califor- of Texas Medical Branch, Galveston,
ain 73029 Texas 55577
TNAK1RHS ANANT (3), Department of -eG JORGEN BROSlUS (41), Fishberg Research
netics, ehT University of Illinois at Chi- Center for Neurobiology, Mount Sinai
cago, Chicago, Illinois 21606 School of Medicine, New York, New York
JANET M. BARSOMIAN (23), New England 92001
Biolabs Inc., Beverly, Massachusetts Z. CAI (10), Department of Immunology,
51910 Mayo Clinic, Rochester, Minnesota
ROBERT L. BEBEE (4), Corporate Research, 50955
GIBCO BRL, Life Technologies Inc., ALLAN CAPLAN (37), Department of Bacte-
Gaithersburg, Maryland 89802 riology and Biochemistry, University of
STEPHAN BECK (15), Imperial Cancer Re- Idaho, Moscow, Idaho 34838
search Fund, London WC2A 3PX, En- C. THOMAS CASKET (7), Howard Hughes
gland Medical Institute, Baylor College of Med-
ASHOK S. BHAGWAT (21), Department of icine, Houston, Texas 03077
Chemistry, Wayne State University, -eD FARID F. CHEHAB (14), Department of Lab-
troit, Missouri 20284 oratory Medicine, University of Califor-
ADI D. BHARUCHA (18, 19), Department of nia, San Francisco, San Francisco, Cali-
Biochemistry, Faculty of Medicine, Laval fornia 34149
University, Ste-Foy, Quebec GIK 7P4, YAWEN L. CHIANG (8), Department oflm-
Canada munology, Genetic Therapy Inc.,
WENDY A. BICKMORE (22), MRC Human Gaithersburg, Maryland 87802
Genetics Unit, Western General Hospital, ING-MING CHIU (44), Departments of later-
Edinburgh EH4 2XU, Scotland hal Medicine and Molecular Genetics,
ADRIAN P. BIRD (22), Institute of Cell and and Comprehensive Cancer Center, ehT
Molecular Biology, University of Edin- Ohio State University, Columbus, Ohio
burgh, Edinburgh EH9 3JR, Scotland 01234
H. C. BIRNBOIM (16), Ottawa Regional BRYAN R. CULLEN (31), Howard Hughes
Cancer Centre, and Departments of Bio- Medical Institute, Section of Genetics,
,.yrtsimehc Medicine, and Microbiology/ Departments of Microbiology and Medi-
Immunology, University of Ottawa, -tO cine, Duke University Medical Center,
tawa, Ontario K1H 8L6, Canada Durham, North Carolina 01772
ix
X CONTRIBUTORS TO VOLUME 216
CRAM DE BLOCK (36), Plant Genetics Sys- ENAITSIRHC GOBLET (17), D~partement de
tems, B-9000 Gent, Belgium Biologie Mol~culaire, Institut Pasteur,
RUDY DEKEYSER (37), Instituut ter Aan- 42757 Paris Cedex ,51 France
moediging van het Wetenschappelijk, -nO JEAN GOULD (30), Soil and Crop Sciences
derzoek in Nijverheid en Landbouw, B- Department, Texas A&M University, Col-
0501 Brussels, Belgium lege Station, Texas 34877
ANASUS ED AL LUNA (33), Centro Nacional SIOqNARF GUIDET (1), P1G Prince de Bre-
de Biotechnologla and Centro de Biologfa tagne Biotechnologie, Penn Ar Prat,
Molecular, Universidad Aut6noma de 05292 St. Pol De Ldon, France
Madrid, Campus de Cantoblanco, 28049 JOHN D. HARDING (4), Corporate Research,
Madrid, Spain GIBCO BRL, Life Technologies Inc.,
NEGR3tJ DENECKE (36), Department of Mo- Gaithersburg, Maryland 89802
lecular Genetics, Swedish University of YRAG G. NOSNAMREH (46), Molecular -eG
Agricultural Sciences, S-75007 Uppsala, netics Laboratory, ehT Salk Institute for
Sweden Biological Studies, San Diego, California
PETER B. DERVAN (27), Arnold and Mabel 83129
Beckman Laboratory of Chemical Syn- PHILIP HIETER (49), Department of Molecu-
thesis, Division of Chemistry and Chemi- lar Biology and Genetics, The Johns
cal Engineering, Pasadena, California Hopkins University School of Medicine,
52119 Baltimore, Maryland 50212
JEFFREY R. ED WET (35), Pfizer Central Re- R. M. HORTON (10), Department of Immu-
search, Pfizer, Inc., Groton, Connecticut nology, Mayo Clinic, Rochester, Minne-
04360 sota 50955
NEELHTAK D'HALLUIN (36), Plant Genetics DENNIS E. HRUBY (32), Center for Gene Re-
Systems, B-9000 Gent, Belgium search and Biotechnology, Department of
SEMAJ EBERWINE (9), Departments of Phar- Microbiology, Oregon State University,
Corvallis, Oregon 13379
macology and Psychiatry, University of
Pennsylvania Medical School, Philadel- JAN SNESSNAJ (36), Plant Genetics Systems,
phia, Pennsylvania 40191 B-9000 Gent, Belgium
GLEN A. EVANS (46), Molecular Genetics Y. W. KAN (14), Department of Laboratory
Laboratory, The Salk Institute for Biolog- Medicine, Howard Hughes Medical Insti-
ical Studies, San Diego, California 83129 tute, University of California, San Fran-
cisco, San Francisco, California 34149
GEORGE R. FEEHERY (23), New England
Biolabs Inc., Beverly, Massachusetts ARDNEJAR P. KANDPAL (5), Department of
51910 Genetics, Yale University School of Medi-
DRAHCIR FINNELL (9), Department of Vet- cine, New Haven, Connecticut 01560
erinary Anatomy~Public Health, Texas DIVAD J. KEMP (12), Menzies School of
A&M University, College Station, Texas Health Research, Casuarina, Northern
34877 Territory 0811, Australia
CARL W. FULLER (29), Research and Devel- SUN CHANG KIM (26), Department of -nO
opment, United States Biochemical Cor- cology, McArdle Laboratory for Cancer
poration, Cleveland, Ohio 22144 Research, University of Wisconsin, Madi-
GULILAT GEBEYEHU (4), Molecular Biology son, Wisconsin 60735
Research and Development, GIBCO MICHAEL KOOB (2, 28), McArdle Labora-
BRL, Life Technologies Inc., Gaithers- tory for Cancer Research, University of
burg, Maryland 89802 Wisconsin, Madison, Wisconsin 60735
CONTRIBUTORS TO VOLUME 216 xi
DAVID LANDRY (23), New England Biolabs MICHAEL NELSON (25), Department of
Inc., Beverly, Massachusetts 51910 Plant Pathology, University of Nebraska,
Lincoln, Nebraska 38586
PETER LANGRIDGE (1), Centre for Cereal
Biotechnology, ehT WRite Agricultural JUAN ORTfN (33), Centro Nacional de
Research Institute, University of Ade- Biotechnologia and Centro de Biologla
laide, Glen Osmond, South Australia Molecular, Universidad Aut6noma ed
,4605 Australia Madrid, Campus de Cantoblanco, 94082
Madrid, Spain
CHENG CHI LEE (7), Institute for Molecular
Genetics, Baylor College of Medicine, MICHAEL PANACCIO (13), Victorian Insti-
Houston, Texas 03077 tute of Animal Science, Attwood, Victoria
,9403 Australia
JAN LEEMANS (36), Plant Genetics Systems,
0009-B Gent, Belgium WILLIAM J. PAVAN (49), Department of Mo-
lecular Biology, Howard Hughes Medical
KIRSTEN LEHTOMA (44), Department of In-
Institute, Princeton University, Prince-
ternal Medicine and Comprehensive Can-
ton, New Jersey 44580
cer Center, ehT Ohio State University,
Columbus, Ohio 01234 L. R. PEASE (10), Department oflmmunol-
ogy, Mayo Clinic, Rochester, Minnesota
GEORGES LI~VESQUE (19), Department of
50955
Biochemistry, Faculty of Medicine, Laval
University, Ste-Foy, Quebec GIK 7P4, I. PELLETIER (40), Institute of Industrial -eG
adanaC netics, University of Stuttgart, D-7000
Stuttgart ,1 Germany
ANDREW M. LEW (13), Walter and Eliza
Hall Institute, Melbourne, Victoria ,0503 SIDNEY PESTKA (20), Department of Molec-
Australia raM Genetics and Microbiology, Univer-
sity of Medicine and Dentistry of New
KENNETH R. LUEHRSEN (35), Department
Jersey, Robert Wood Johnson Medical
of Biological Sciences, Stanford Univer-
School, Piscataway, New Jersey 45880
sity, Stanford, California 50349
JAMES C. PIERCE (47), Cancer Therapeutic
SCOTT MACKLER (9), Department of Phar-
Program, ehT Du Pont Merck Pharma-
macology, University of Pennsylvania
ceutical Company, Wilmington, Dela-
Medical School, Philadelphia, Pennsylva-
ware 08891
ain 40191
ANNA J. PODHAJSKA (26), Department of
MICHAEL H. MALIM (31), Howard Hughes
Microbiology, University of Gdansk,
Medical Institute, Departments of Micro-
222-08 Gdansk, Poland
biology and Medicine, University of
Pennsylvania School of Medicine, Phila- MATTHEW L. POULIN (44), Department
delphia, Pennsylvania 40191 Molecular Genetics, The Ohio State Uni-
versity, Columbus, Ohio 01234
MICHAEL MCCLELLAND (25), Department
of Plant Pathology, University of Ne- EDOUARD PROST (17), D~partement de
braska, Lincoln, Nebraska 38586 Biologie Mol~culaire, Institut Pasteur,
42757 Paris Cedex ,51 France
KEVIN MIYASHIRO (9), Department of Phar-
macology, University of Pennsylvania J. K. PULLEN (10), Department oflmmunol-
Medical School, Philadelphia, Pennsylva- ogy, Mayo Clinic, Rochester, Minnesota
ain 40191 50955
DONALD T. MOIR (50), Department of Hu- PETER J. PUNT (39), Department of Molecu-
man Genetics and Molecular Biology, lar Genetics and Gene Technology, Medi-
Collaborative Research, Inc., Waltham, cal Biological Laboratory, 2280 AA
Massachusetts 45120 Rijsw(jk, The Netherlands
xii CONTRIBUTORS TO VOLUME 216
ROGER H. REEVES (49), Department of WACLAW IKSLABYZS (2, 26), McArdle Lab-
Physiology, The Johns Hopkins Univer- oratory for Cancer Research, University
sity School of Medicine, Baltimore, Mary- of Wisconsin, Madison, Wisconsin 60735
land 50212
KENNETH D. TARTOF (48), Institute for
ARLETTE STREANYER (36), Plant Genetics Cancer Research, Fox Chase Cancer
Systems, B-9000 Gent, Belgium Center, Philadelphia, Pennsylvania 11191
DAVID RON (34), Laboratory of Molecular HIROO TOYODA (ll), Medical Genetics-
Endrocrinology, Massachusetts General Birth Defects Center, Department of
Hospital, Boston, Massachusetts 41120 Medicine and Pediatrics, Cedars-Sinai
J. M. SHORT (42, 43), Strategene Cloning Medical Center, UCLA School of Medi-
Systems, La Jolla, California 73029 cine, Los Angeles, California 84009
SALGUOD R. SMITH (50), Department of LEVY ULANOVSKY (6), Department of
Human Genetics and Molecular Biology, Structural Biology, The Weizmann Insti-
Collaborative Research, Inc., Waltham, tute of Science, Rehovot ,00167 Israel
Massachusetts 45120 CRAM VAN MONTAGU (37), Laboratorium
ADRIENNE P. SMYTH (50), Department of voor Genetica, Universiteit Gent, 0009-B
Human Genetics and Molecular Biology, Gent, Belgium
Collaborative Research, Inc., Waltham, CEES A. M. J. J. NAV NED HONDEL (39),
Massachusetts 45120 Department of Molecular Genetics and
KEN SNIDER (46), Molecular Genetics Lab- Gene Technology, Medical Biological
oratory, The Salk Institute for Biological Laboratory, 2280 AA Rijswijk, ehT Neth-
Studies, San Diego, California 83129 erlands
GOMEAJ HOS (20), Department of Molecular M. R. VEN MURTHY (18, 19), Department of
Genetics and Microbiology, University of Biochemistry, Faculty of Medicine, Laval
Medicine and Dentistry of New Jersey, University, Ste-Foy, Quebec GIK 7P4,
Robert Wood Johnson Medical School, Canada
Piscataway, New Jersey 45880 P. VIELL (40), Institute of lndustrial Genet-
ics, University of Stuttgart, D-7000 Stutt-
J. A. SORGE (42, 43), Strategene Cloning
gart ,1 Germany
Systems, La Jolla, California 73029
VIRGINIA WALBOT (35), Department of Bio-
ENNIROC RECNEPS (9), Department of Phar-
logical Sciences, Stanford University,
macology, University of Pennsylvania
Stanford California 50349
Medical School, Philadelphia, Pennsylva-
nia 40191 JEFF WALL (14), Department of Laboratory
Medicine, University of California, San
NAT L. GREBNRETS (47), Cancer Therapeu-
Francisco, San Francisco, California
tic Program, The Du Pont Merck Phar-
34149
maceutical Company, Wilmington, Dela-
ware 08891 DAVID C. WARD (5), Department of Genet-
ics, Yale University School of Medicine,
TTOCS A. LEBORTS (27), Arnold and Mabel
New Haven, Connecticut 01560
Beckman Laboratory of Chemical Syn-
thesis, Division of Chemistry and Chemi- NAMREHS M. NAMSSIEW (5), Department of
cal Engineering, Pasadena, California Genetics, Yale University School of Medi-
52119 cine, New Haven, Connecticut 01560
KIRANUR N. SUBRAMANIAN (3), Depart- TREBOR G. WHALEN (17), D~partement de
ment of Genetics, The University of Illi- Biologie Mol~culaire, Institut Pasteur,
nois at Chicago, Chicago, Illinois 21606 42757 Paris Cedex ,51 France
CONTRIBUTORS TO VOLUME 216 xiii
ELIZABETH M. WILSON (32), Center for LEAHCIM WITTY (38), Department of Plant
Gene Research and Biotechnology, De- Sciences, University of Cambridge, Cam-
partment of Microbiology, Oregon bridge 2BC 3EA, England
State University, Corvallis, Oregon ANDREW O. ZELENETZ (45), Division of
13379 Hematologic Oncology/Lymphoma of
Memorial Hospital Program ni Molecular
YERFFOEG G. WILSON (23, 24), New En- Biology of the Sloan-Kettering Institute,
gland Biolabs Inc., Beverly, Massachu- Memorial Sloan-Kettering Cancer Cen-
setts 51910 ter, New York, New York 12001
1 ESABAGEM DNA MORF STNALP 3
1 Megabase DNA Preparation from Plant Tissue
By SIOqNARF GUIDET and PETER EGDIRGNAL
Introduction
Traditional DNA extraction methods yield fragments of about 50 to
001 kilobase pairs (kbp) in length. The largest DNA fragments that can be
separated by conventional electrophoresis in an agarose gel are 30 to
40 kbp in size. In contrast, the pulsed-field gel electrophoresis (PFGE)
technique allows the separation of DNAs of more than 10,000 kbp (10
Mbp). The different principles involved in PFGE are represented by vari-
ous acronyms such as FIGE, OFAGE, TAFE, and CHEF. All involve
repeated reorientation of the DNA molecules inside the gel matrix due
to corresponding changes in electric field parameters (electrode angle,
switching time, field inversion, etc.; for a review see Ref. .)1 To date the
fractionation of DNA molecules has been extended to 21 Mbp, 2 but there
does not seem to be any theoretical limit.
To take advantage of these dramatic improvements biologists have
designed methods to prepare high molecular weight DNA molecules, so-
called megabase DNA (Mbp DNA). 3 For various reasons, plant molecular
biologists have been slow to develop specific protocols suitable for prepar-
ing Mbp DNA. Without exception, the methods used to prepare plant Mbp
DNA involved the preparation of protoplasts as a preliminary step, that
is, plant cells are freed of their cell wall by digestion with specific en-
zymes. 8-4 To circumvent this tedious task, we designed an alternative
method that appeared to be both rapid and efficient. 9 The description of
an updated version of this method is the subject of the present article.
Principle of Method
The principle of the method is straightforward. It is based on the
assumption that grinding leaf tissue in the presence of liquid nitrogen with
t R. Anand, Trends Genet. 2, 278 (1986).
: M. J. Orbach, D. Vollrath, R. W. Davis, and C. Yanofsky, Mol. Cell. Biol. 8, 1469 (1988).
3 D. C. Schwartz and C. R. Cantor, Cell (Cambridge, Mass.) 37, 76 0984).
4 p. Guzman and J. R. Ecker, Nucleic Acids Res. 16, ll091 (1988).
5 M. W. Ganal, N. D. Young, and S. D. Tanksley, Mol. Gen. Genet. 215, 395 (1989).
6 C. Jung, M. Kleine, F. Fischer, and R. G. Herrmann, Theor. Appl. Genet. 79, 663 0990).
7 R. A. J. van Daelen, J. J. Jonkers, and P. Zabel, Plant Mol. Biol. 12, 341 (1989).
8 W. Y. Cheung and M. D. Gale, Plant Mol. Biol. 14, 881 (1990).
9 F. Guidet, P. Rogowsky, and P. Langridge, Nucleic Acids Res. 18, 4955 (1990).
thgirypoC © 2991 by cimedacA Press, Inc.
SDOHTEM NI ,YGOLOMYZNE VOL. 612 llA rights of noitcudorper ni any form reserved.
4 ISOLATION, SYNTHESIS, DETECTION OF DNA AND RNA [1]
a mortar and pestle allows the preparation of plant cells, either isolated or
in small aggregates. These plant cells, surrounded by a more or less
damaged cell wall, contain intact organelles and their membranes are
amenable to digestion by the combined action of a detergent (sarkosyl) and
a proteolytic enzyme (proteinase K). The integrity of the DNA molecules is
maintained by the addition of a chelating agent (ethylenediaminetetraacetic
acid; EDTA), which helps protect the DNA from nucleases. Most impor-
tant, the plant material (powder) is embedded in agarose prior to digestion,
thus avoiding any mechanical shearing during subsequent treatments.
Once the various cell membranes have been dissolved, the DNA molecules
are liberated from their associated proteins by the proteinase K. The entire
treatment is done at 53-55 °, which is still within the optimal temperature
range of action for the proteinase K but well out of the active range for
most plant nucleases. The DNA remains in the cavities created inside the
agarose plugs by the original plant cells while solutes and small products
of cell wall degradation diffuse out of the plugs. The DNA is still accessible
to DNA-modifying enzymes such as restriction endonucleases and can be
subjected to molecular biological manipulation.
Materials and Reagents
The plant materials used are either green leaves of 10-day-old seedlings,
seeds, or commercial flour. Wheat-rye recombinant plants have been
described in Rogowsky et al.l° and are obtained from Ken W. Shepherd
(Waite Institute, South Australia). Seeds from alfalfa, lentils, and soybeans
are from a local shop. Rye (cv. 'South Australian') flour is from W. Thomas
Company (Port Adelaide, South Australia).
Low melting temperature (LMP) and LE agarose are both from FMC
BioProducts (Rockland, ME), proteinase K and restriction enzymes are
from Boehringer (Mannheim, Germany), and radiolabeled dCTP and the
transfer membrane HyBond N + are from Amersham (Arlington Heights,
IL). The PFGE system used was a CHEF DR II from Bio-Rad Labora-
tories (Richmond, CA).
Solutions used to treat or store the plugs include a lysis solution (10
mM Tris-HC1, pH 8.0,500 mM EDTA, 1% (v/v) sarkosyl, 1 mg/ml protein-
ase K), × 1 ET 1( mM Tris-HCl, pH 8.0, 50 mM EDTA), and TE (10 mM
Tris-HC1, pH 8.0, l mM EDTA).
After the electrophoretic runs the gels are stained with ethidium bro-
mide (1/zg/ml) for 45 min and destained extensively to optimize the signal-
0i p. Rogowsky, F. Guidet, P. Langridge, K. W. Shepherd, and R. M. D. Koebner, Theor.
Appl. Genet. 82, 735 (1991).
[1] MEGABASE DNA FROM PLANTS 5
to-background ratio. Destaining of up to 02 hr does not visibly affect the
sharpness of the DNA bands. The gels are then photographed and irradi-
ated for 1 min with 254-nm UV light, depurinated in 0.25 M 1CH for 51
min, treated with alkali (1.5 M NaC1, 0.5 M NaOH) twice for 51 min, and
equilibrated in the alkali transfer solution (I 5. M NaC1, 0.25 M NaOH) for
51 min prior to setting up the capillary transfer system. The transfer lasts
for 42 hr, The hybridization conditions have been reported in Rogowsky
et al. °1 The probe pAWI73 detects a moderately repeated rye-specific
element. 1
Method
About 0.4 g of young green leaves are ground to a fine powder in liquid
nitrogen using a pestle and mortar. The powder is transferred to a crucible
preheated to ,° 05 mixed with 2 ml of 0.7% (w/v) LMP agarose in x 1 ET,
and gently stirred with a sterile spatula to obtain a homogeneous mixture
(alternatively the powder can be mixed with I ml of I x ET and then added
to 1 ml of 1.4% agarose solution). The mixture is then poured directly into
the mold (Bio-Rad CHEF DR II mold), shaking it gently while pouring to
maintain the homogeneity of the mixture. It is then allowed to set at ° 4 for
02 min in a lying positiont o avoid a deposit of debris at the bottom of each
agarose plug.
The plugs are transferred into petri dishes and incubated in the lysis
solution; we use 01 ml of lysis solution per l0 plugs (each plug is about
250/A agarose mixture). The incubation is done at 53-55 ° on a rocking
platform in an oven or by floating the petri dishes in a water bath (to be
on the safe side it is wise to float the petri dishes inside a plastic box with
a minimum of water, the box itself floating in the water bath). At the end
of the treatment the plugs are stored at ° 4 in x 1 ET.
We have extended the method and used flour or crushed seeds instead
of the leaf material. 21 The seeds are crushed in a mortar and pestle without
liquid nitrogen. Most of the results presented here have been obtained by
using crushed seeds or flour.
Results
Source of Material
The present method of direct Mbp DNA isolation was developed be-
cause high yields were obtained rapidly and without the elaborate tech-
tI F. Guidet, P. Rogowsky, C. Taylor, W. Song, and P. Langridge, Genome 34, 18 (1991).
21 F. Guider and P. Langridge, C.R. Acad. Sci. Paris, Ser. 3 314, 7 (1992).
6 ISOLATION, SYNTHESIS, DETECTION OF DNA AND RNA ]1[
SIZE M 0 1 2 4 7 2448
(Kbp)
2500
1600
1125
1020
945
O58
OO8
770
OO7
O36
580
460
370
290
245
A
Fi6. .1 Release and restriction digestion of DNA from rye flour embedded in agarose.
Two series of plugs were incubated with lysis solution at 55 ° for the periods indicated at the
top of each lane (time is in hours). One series )A( was electrophoresed directly (run condi-
tions: time ramp 05 to 09 sec at 002 V, in a %1 agarose gel in 5.0 × TBE buffer for 42 hr).
The other series was incubated with HindIII and subsequently electrophoresed )B( (run
conditions: time ramp 1 to 6 sec at 521 V, in a agarose 1.5% gel in 5.0 × TBE buffer for 81
hr). The arrow in )B( indicates relic DNA that hybridizes with an rDNA probe. The from gel
)B( was transferred and probed with pAW 371 (C). )A( and )B( are ethidium bromide-stained
gels; )C( is an autoradiogram.
niques involved in the preparation of nuclei or protoplasts. Like other
authors 7,6 we were not able to isolate Mbp DNA from nuclei, although we
obtained good-quality DNA from protoplasts prepared from young leaves
or suspension cultures. However, the yield of leaf protoplasts, especially
in the case of cereals, is very low; that is, only a small fraction of the
leaf cells can be turned into protoplasts and are amenable to lysis. Cell