Pulsed Field Gel Electrophoresis. A Practical Guide

254 Pages·1993·5.836 MB·English

by  Bruce Birren and Eric Lai (Auth.)

PULSED FIELD GEL
ELECTROPHORESIS
A Practical Guide j
BRUCE BIRREN
Division of Biology
California institute of Technology
Pasadena, California
ERIC LAI
Department of Pharmacology
University of North Carolina
Chapel Hill, North Carolina
ACADEMIC PRESS, INC.
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Cover photograph is courtesy of Yoshiaki Tachi-iri,
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Copyright © 1993 by ACADEMIC PRESS, INC.
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Pulsed Field Gel Electrophoresis: A Practical Guide
by Bruce Β irren, Eric Lai
International Standard Book Number: 0-12-101290-5
PRINTED IN THE UNITED STATES OF AMERICA
93 94 95 96 97 98 EB 9 8 7 6 5 4 3 2 1
To the Memory of My Mother, Wah Leung
Eric Lai
PREFACE
The ability to separate very large DNA molecules by pulsed field gel electro-
phoresis (PFGE) instantly changed the scope of problems amenable to molecu-
lar approaches. With the codevelopment of methods for preparing and analyz-
ing large DNA molecules, pulsed field gels have precipitated explosive
progress in a number of diverse disciplines. In many cases, for example, in
microbial genetics or crop sciences, the techniques of PFGE represent a radical
departure from traditional methods. In addition, the rapid evolution of early
pulsed field hardware repeatedly made certain procedures or equipment obso-
lete. Now, although commercial apparatuses offer consistent performance,
one must choose between a number of very different devices. Thus, we have
attempted to provide a complete guide that will serve as an introduction to the
equipment, the process, and the procedures used for performing pulsed field
gel electrophoresis. We have attempted to accommodate researchers working
with a variety of organisms, and with different levels of interest in the process
itself.
We have been involved in the development of the instrumentation, pro-
cedures, and applications of pulsed field gel technology since the early days of
home-designed and homemade equipment. When we began, desperation or a
love of electronics were the prime motivations for undertaking pulsed field
experiments. With the advent of commercially available equipment and mark-
ers, the field has become less of an art and more of a science, offering routine
and reproducible separations. It was not until we began teaching PFGE in
courses and workshops that we realized the need for a comprehensive manual.
This is the first book to provide details on all the aspects of PFGE, from
selection and setup of the equipment, to preparation of samples and selec-
tion of separation conditions, to the many special applications of pulsed field
gels.
xvii
xviii PREFACE
Although this book serves as an introduction to PFGE to researchers who
are new to the field, we have included information that will aid experienced
users who would like to learn more about pulsed field technology and how best
to exploit it. Thus, the procedures include both step-by-step instructions and
notes that discuss additional aspects or alternatives to the techniques pre-
sented. In addition, many of the tables are intended as a reference source for
users long after they have become comfortable running pulsed field gels. We
have also assembled an extensive bibliography, referenced by topic and organ-
ism. This offers a view of what has, and has not, been accomplished with pulsed
field gels and will stimulate further exploration of topics beyond the scope of
this book.
We are deeply indebted to our wives and children for their ongoing
patience and encouragement. We are pleased to thank Leroy Hood and Melvin
Simon for creating the opportunity for our work together, Valeta Gregg for
initiating this project, and Phyllis Moses for having the stamina to see it
through. We thank all our colleagues over the years who have commented on
our evolving protocols, as well as Catherine Esnault, Celeste Cantrell, Lee Hui,
and Jeff Stein for proofreading parts of the manuscript. We are especially
grateful to Joan Kobori for her numerous suggestions and improvements to
the entire manuscript. Finally, we would like to thank Gilbert Chu and Ted
Davis for contributing their blueprints and data; all the companies that have
provided information; and those who have contributed procedures, figures,
and tables.
Bruce Birren
Eric Lai
C H A P T ER
1
INTRODUCTION TO CONVENTIONAL AND
PULSED FIELD GEL ELECTROPHORESIS
1.1 Conventional Gel Electrophoresis
Conventional gel electrophoresis of DNA molecules is carried out by placing
DNA samples in a solid matrix (most commonly agarose or Polyacrylamide)
and inducing the molecules to migrate through the gel under a static electric
field. In the absence of external forces, DNA molecules exist in a relaxed form;
their movement is mostly Brownian motion (Fig. 1.1 A). Under the influence
of an electric field (Ej), the DNA molecules elongate and align with the field
and migrate toward the anode by a process termed "reptation" (Fig. LIB).
Reptation of DNA through the gel can be likened to the movement of a snake
through bamboo: the head selects the path and the rest of the molecule follows.
The separation of molecules of different size predominantly depends on the
sieving properties of the gel matrix; smaller fragments can move more easily
through the solid matrix. Several parameters affect the separation and mobility
of DNA molecules in gel electrophoresis, including composition and concentra-
tion of the gel, the buffer, the temperature, and the voltage gradient of the
electric field. Using special conditions, static field gel electrophoreis can sepa-
rate DNA molecules as large as 50 kilobase pairs (kb) (represented by the
medium-size molecule in Fig. 1.1 A). However, in ordinary situations, all mole-
cules larger than 20 kb will show essentially the same mobility in a static electric
field and, thus, will not be separated from each other (Fig. LIB), because DNA
molecules greater than 20 kb (largest molecule in Fig. LIA) will have the same
cross-sectional area after they align with the electric field. Early attempts to
extend the range of separation to larger molecules relied on reducing the
agarose concentration to as low as 0.1% and using very low voltage gradients.
1
2 1. INTRODUCTION TO CONVENTIONAL AND PULSED FIELD GEL ELECTROPHORESIS
Figure 1.1 Schematic illustration of DNA separation in conventional and pulsed field gel electro-
phoresis. (A) DNA of various sizes exists as a random coil in the absence of an external electric
field. Assume the sizes of the DNA molecules represented to be 5, 50, and 500 kb (from top to
bottom). (B) Separation of DNA molecules in conventional gel electrophoresis with a static electric
field Ε1. All molecules are aligned with the field, but the 50 and 500 kb molecule s present essentially
the same cross sectional area in the gel and thus migrate at the same rate. (C) In a pulsed field
gel, the first electric field (El in panel B) is turned off and a second field (E2) is activated in a new
direction. Shortly after the new field is applied, the smallest fragment has realigned in the direction
of the new field, while the large molecules have yet to reorient. The arrows show the possibl e paths
the larger DNAs must choose between before they can migrate in the direction of the second
electric field.
Unfortunately, low-percentage agarose gels are mechanicall y very difficult to
handle and the use of low voltage gradients requires run times of days to weeks.
Even under these extreme conditions, separation of DNA molecules larger
than a few hundred kilobase pairs does not seem possible.
1.2 Pulsed Field Gel Electrophoresis
In the early 1970s, Bruno Zimm and co-workers (Klotz and Zimm, 1972)
showed that, after the removal of an electric field, the elongated DNA mole-
cules relax back to their unperturbed state; the rate of relaxation is dependent
on the length of the DNAs. David Schwartz, then a student of Zimm, attempted
to exploit this size-dependent relaxation to separate large DNA molecules.
Periodically changing the orientation of the electric field would force the DNA
molecules in the gel to relax on removal of the first field and elongate to align
1.3 TERMS THAT HAVE BEEN USED IN PULSED FIELD GEL ELECTROPHORESIS 3
with the new field. This process should be size dependent. In the laboratory
of Charles Cantor, Schwartz demonstrated the effectiveness of field switching
by separating yeast chromosomes several hundred kilobases in length.
The principle of pulsed field gel electrophoresis (PFGE) separation fol-
lows. When the first electric field (Ej) is applied to the gel, as in Fig. LIB, DNA
molecules elongate in the direction of the field and begin to migrate in the gel.
The first electric field is then removed and a second field (E ), at some angle
2
to the first field, is activated (Fig. 1.1C). The DNA must change conformation
and reorient before it can migrate in the direction of the second electric field.
The time required for this reorientation has been found to be very sensitive to
the length of the molecule (i.e., molecular weight). Larger DNA molecules
take more time to realign after the field is switched than smaller ones do
because of the physical barrier of the agarose matrix. Hence, molecules of
increasing size must spend a larger portion of each switching cycle reorienting
before they can begin to migrate through the gel. As long as the alternating
fields are equal with respect to length and voltage, the DNA will migrate in a
straight path down the gel that reflects the sum of the many short zig-zag steps
actually taken.
1.3 Terms That Have Been Used in Pulsed Field
Gel Electrophoresis
Because of the additional electric fields involved in PFGE, a number of terms
have been introduced to describe fully the electrophoretic conditions used.
Table 1.1 lists terms commonly used in PFGE.
TABLE 1.1
Terms Used in PFGE
Pulsed field Any electrophoresis process that uses more than one electric field in which
the electric fields are activated alternatingly
Switch interval Amount of time each of the alternating electric fields is active (also referred
to as switch time, pulse time)
Reorientation angle Acute angle between two alternating electric fields (i.e., the angle between
the different directions that the DNA molecules will migrate)
Field inversion PFGE system in which the two alternating fields are oriented opposite each
other, that is, a reorientation angle of 180°
Voltage gradient Electrical potential applied to the gel, measured in volts per centimeter
Homogeneous field Electric field that has uniform potential differences across the whole field
4 1. INTRODUCTION TO CONVENTIONAL AND PULSED FIELD GEL ELECTROPHORESIS
1.4 Microscopic Observations of DNA Molecules in Pulsed
Field Gel Electrophoresis
The effectiveness of pulsed field gels was immediately obvious from the first
photographs published by Schwartz and Cantor (1984). However, the process
by which the DNA reorient s with the switching fields and, thus, the actual
basis for the separation remained a matter of speculation for years, despite
numerous biophysical and theoretical studies. Finally, direct microscopic obser-
vation of individual DNA molecules in the gel provided clear pictures of how
large DNA molecules move and change direction during PFGE. Figure 1.2
shows the movement of a single fluorescently stained phage T2 DNA molecule
(164 kb) that is undergoing PFGE on a microscope slide. The DNA molecule
is stretched by the initial electric field (oriented horizontally from left to right)
(Fig. 1.2A). The first electric field then is turned off and replaced by a second
electric field (oriented vertically from top to bottom) (Fig. 1.2B—G). As can be
seen in these photographs, the molecule forms kinks (white arrows) as it
attempts to align with the second electric field. The different kinks compete to
become the new "head" that will lead the migration of the molecule (Fig.
1.2C-F). One of the kinks eventually wins, and the molecule migrates in the
new direction. The longer the DNA molecule, the more kinks are formed and
Figure 1.2 Time-lapsed photographs of a T2 DNA molecule undergoin g pulsed field gel electro-
phoresis PFGE. (A) The molecule aligned along the horizontal direction according to the first electric
field. The first electric field is then turned off and the second field (from top to bottom) is activated.
Bar = 4 micron. Kinks begin to appear in Β and C, and the kinks compete with each other, until
finally, the right end of the molecule becomes the leading end and pulls the molecule to migrate
in the second field. (Reprinted with permission from Gurrier: et al., copyright © 1990, American
Chemical Society.)
PULSED FIELD GEL ELECTROPHORESIS NOMENCLATURE 5
the longer establishment of a new head and migration in the new direction
takes. Research such as the microscope observations have advanced our under-
standing of the basic concept of PFGE. The precise rules that govern which
path a DNA molecule will take when the field switches remain unclear. Com-
puter modeling of the process has been able to reproduce some of the phenom-
ena associated with PFGE.
5 Pulsed Field Gel Electrophoresis Nomenclature
PFGE nomenclature remains confusing because of the large number of acro-
nyms used over the years (Table 1.2). The original term pulsed field gradient
gel electrophoresis (PFGE) was applied by Schwartz and Cantor (1984) to any
gel run using alternating multiple electric fields. It is now clear that a field
gradient is neither an important nor a desirable aspect of pulsed field gels.
The abbreviation PFG is now taken to mean pulsed field gel, not pulsed field
gradient. Subsequently, other names (e.g., OFAGE, FIGE, TAFE, CHEF,
PACE, RGE, crossed-field electrophoresis, ZIFE, ST/RIDE) have been given
to pulsed field electrophoresis systems that involve variations on the original
electrode geometry, homogeneity, and method of reorientation of the electric
fields (see Chapter 2). Most of these names are used to describe a particular
hardware design (e.g., electrode geometry, electrical circuit), not a specific
TABLE 1.2
Pulsed Field Gel Acronyms
Acronym Electrophoresis systems Reference
PFGE Pulsed field gradient gel electrophoresis Schwartz and Cantor (1984)
OFAGE Orthogonal field alternation gel electrophoresis Carle and Olson (1984)
TAFE Transverse alternating field electrophoresis Gardiner et al. (1986)
FIGE Field inversion gel electrophoresis Carle et al. (1986)
CHEF Contour clamped homogeneous electric field Chu et al. (1986)
RGE Rotating gel electrophoresis Serwer (1987)
Crossed-field gel electrophoresis Southern et al. (1987)
Rotaphor Rotating electrodes gel electrophoresis Biometra (see Table 2.3)
Waltzer Crossed field gel electrophoresis Anand et al. (1989)
PACE Programmable autonomously controlled Clarke al. (1988)
electrodes
ZIFE Zero integrated field electrophoresis Turmel et al. (1990)
ST/RIDE Simultaneous tangential/rectangular inversion Kolble and Sim (1991)
decussate electrophoresis