Table Of ContentCHAIVER 1
DNA Sequencing
Hugh G. Grifin and Annette M. Grifin
1. Introduction
Methods to determine the sequenceo f DNA were developed in the
late 1970s (1,2) and have revolutionized the science of molecular
genetics. The DNA sequences of many different genes from diverse
sources have been determined, and the information is stored in interna-
tional databanks such as EMBL, GenBank, and DDBJ. Many scientists
now accept that sequence analysis will provide an increasingly use-
ful approach to the characterization of biological systems. Projects are
already underway to map and sequencet he entire genome of organisms
such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis
elegans, and Homo sapiens. In the recent past, large-scale sequenc-
ing projects such as these were often dismissed as prohibitively expen-
sive and of little short-term benefit, while DNA sequencing itself was
seen as a repetitive and unintellectual pursuit. However, this view is
now changing and most scientists recognize the importance of DNA
sequence data and perceive DNA sequencing as a valuable and often
indispensable aspect of their work. Recent technological advances,
especially in the area of automated sequencing, have removed much
of the drudgery that used to be associated with the technique, and
modern innovative computer software has greatly simplified the analy-
sis and manipulation of sequence data. Large-scale sequencing
From Methods m Molecular Biology, Vol. 23. DNA Sequencmg Protocols
Edited by. H. and A. Gnffm Copyright Q1993 Humana Press Inc., Totowa, NJ
1
2 Griffin and Griffin
projects, such as the Human Genome Project, produce the DNA sequen-
ces of many unknown genes. Such data provide an impetus for molec-
ular biologists to apply the techniques of reverse genetics to produce
probes and antibodies that can be used to identify the gene product, its
cellular location, and its time of appearancei n the developing cell (3).
A function can be assigned by mutant analysis or by comparison of
the deduced amino acid sequence with proteins of known function.
Therefore, DNA sequencing can act as a catalyst to stimulate future
research into many diverse areas of science.
The two original methods of DNA sequencingd escribed in 1977 (1,2)
differ considerably in principle. The enzymatic (or dideoxy chain termina-
tion) method of Sanger (I) involves the synthesis of a DNA strand from
a single-stranded template by a DNA polymerase. The Maxam and
Gilbert (or chemical degradation) method (2) involves chemical degra-
dation of the original DNA. Both methods produce populations of radioac-
tively labeled polynucleotides that begin from a fixed point and terminate
at points dependent on the location of a particular base in the original
DNA strand. The polynucleotides are separated by polyacrylamide
gel electrophoresis, and the order of nucleotides in the original DNA
can be read directly from an autoradiograph of the gel (4).
Although both techniques are still used today, there have been many
changes and improvements to the original methods. While the chemi-
cal degradation method is still in use, the enzymatic chain termina-
tion method is by far the most popular and widely used technique for
sequence determination. This process has been automated by utilizing
fluorescent labeling instead of radioactive labeling (Chapters 33-37),
and the concepts of polymerase chain reaction (PCR) technology have
been harnessed to enable the sequencing reaction to be “cycled” (Chap-
ters 21, 26, and 34). Other recent innovations include multiplexing
(Chapter 28), sequencing by chemiluminescence rather than radioac-
tivity (Chapter 29), solid phases equencing (Chapter 25), and the use of
robotic work stations to automate sample preparation and sequenc-
ing reactions (Chapter 38).
2. Maxam and Gilbert Method
In the original Maxam and Gilbert method (2) a fragment of DNA
is radiolabeled at one end and then partially cleaved in four different
chemical reactions, each of which is specific for a particular base or
DNA Sequencing 3
type of base. This results in four populations of labeled polynucleo-
tides. Each radiolabeled molecule extends from a fixed point (the
radiolabeled end) to the site of chemical cleavage, which is determined
by the DNA sequence of the original fragment. Since the cleavage is
only partial, each population consists of a mixture of molecules, the
lengths of which are determined by the base composition of the origi-
nal DNA fragment. The four reactions are electrophoresed in adjacent
lanes through a polyacrylamide gel. The DNA sequence can then be
determined directly from an autoradiograph of the gel. The original
method has been improved over the years (5). Additional chemical
cleavage reactions have been devised (6), new end-labeling techniques
developed (7,8), and shorter, simplified protocols have been produced
(Chapter 32). The main advantage of chemical degradation sequenc-
ing is that sequence is obtained from the original DNA molecule and
not from an enzymatic copy. It is therefore possible to analyze DNA
modifications such as methylation, and to study protein/DNA interac-
tions. Chemical sequencing also enables the determination of the DNA
sequence of synthetic oligonucleotides. However, the Sanger method
is both quicker and easier to perform and must remain the method of
choice for most sequencing applications.
3, Sanger Method
The Sanger (or chain termination) method (I) involves the synthe-
sis of a DNA strand from a single-stranded template by a DNA poly-
merase. The method depends on the fact that dideoxynucleotides
(ddNTPs) are incorporated into the growing strand in the same way
as the conventional deoxynucleotides (dNTPs). However, ddNTPs
differ from dNTPs becauset hey lack the 3’-OH group necessaryf or chain
elongation. When a ddNTP is incorporated into the new strand, the
absence of the hydroxyl group prevents formation of a phosphodiester
bond with the succeeding dNTP and chain elongation terminates at
that position. By using the correct ratio of the four conventional dNTPs
and one of the four ddNTPs in a reaction with DNA polymerase, a
population of polynucleotide chains of varying lengths is produced.
Synthesis is initiated at the position where an oligonucleotide primer
anneals to the template, and each chain is terminated at a specific
base (either A, C, G, or T depending on which ddNTP was used). By
using the four different ddNTPs in four separate reactions the com-
4 Griffin and Griffin
plete sequence information can be obtained. One of the dNTPs is
usually radioactively labeled so that the information gained by elec-
trophoresing the four reactions in adjacent tracks of a polyacryla-
mide gel can be visualized on an autoradiograph.
The original method used the Klenow fragment of DNA polymerase
I to synthesize the new strands in the sequencing reactions, and this
enzyme is still used today (Chapter 12). Other enzymes such as
Sequenase (Chapter 14), T7 polymerase (Chapter 13), and Taq poly-
merase (Chapter 15) are also widely used. Each enzyme has its own
particular properties and qualities, and the choice of polymerase will
depend on the type of template and the sequencing strategy employed.
4. Templates for DNA Sequencing
The polymerase reaction requires single-stranded template. This is
usually achieved by utilizing Ml3 phage that can produce large
amounts of just one strand of DNA as part of its normal replicative
cycle. Double stranded (replicative form) Ml3 can also be isolated, and
this is used to clone the DNA fragment to be sequenced. The qualrty
of DNA sequenced ata achieved using Ml3 template is extremely good
and many researchers prefer to subclone to Ml3 prior to sequencing.
Sequencing reactions can be performed directly on plasmid DNA,
the double stranded molecule being denatured prior to sequencing.
Recent innovations in DNA purification techniques and the availability
of improved polymerases have greatly enhanced the quality of data
produced by plasmid sequencing methods (Chapters 14, 18, and 19).
Sequence determination can also be performed directly on cosmid
clones (Chapter 21), lambda clones (Chapter 20), and on PCR prod-
ucts (Chapters 23-25). In particular, the advento f cycle sequencing( Chap-
ter 26) has vastly increased the range of templates that can be used.
5. Sequencing Strategies
A lot of sequencing performed is confirmatory sequencing to check
the orientation or the structure of newly constructed plasmids, or to
determine the sequence of mutants. This type of sequencing can be
easily achieved by subcloning a restriction fragment into Ml3 and
sequencing using the universal primer. Alternatively, a custom-designed
oligonucleotide primer can be synthesized and sequencing performed
without the need for any subcloning.
DNA Sequencing 5
The determination of long tracts of unknown sequence, however,
requires careful planning and the utilization of one of a variety of strate-
gies including: the shotgun approach, directed sequencing strategies,
and the gene walking technique. A random, or shotgun, approach
involves subcloning random fragments of the target DNA to an appro-
priate vector such as Ml3 (Chapter 7). Sequencesf rom these recombi-
nants are determined at random until the individual readings can be
assembled into a contiguous sequence. This is achieved using a
sequence assembly computer program (9,lO). The disadvantage of
this method is the redundancy in the sequence data obtained, each
section of DNA being sequenced several times over. However, the
strategy benefits from making no prior assumptions about the DNA
to be sequenced, such as base composition or the presence of certain
restriction sites.
Directed strategies usually involve the construction of a nested set
of deletions of the fragment to be sequenced. Progressive deletions
of the fragment are generatedw ith a nuclease,e achd eletion being approx-
imately 200-300 bp. Following deletion the fragments are recloned
into Ml3 or a plasmid vector adjacent to the universal primer site.
The subclones are then sequenced in order of size, with the sequence
of each clone overlapping slightly with the one before. In this way, a
large tract of contiguous sequencei s determined on one gel. The disad-
vantage is the labor and time involved in constructing the deletions.
Several methods are available for deletion construction including the
use of exonuclease III (Chapter 8), T4 DNA polymerase (Chapter 9),
and DNase I (Chapter 10). It is essential to sequence both strands of
the DNA and this usually entails generating two sets of deletions.
Perhaps the simplest method of sequencing is the gene walking
technique (Chapter 11). This involves the initial sequencing of approx-
imately 200-400 bp of the end of a cloned fragment using the univer-
sal primer (the sequence of the other end can be achieved with the
reverse primer). This sequence information is then used to design a
new oligonucleotide primer, which will provide the sequence of the
next 200-400 bp, and so on across the entire length of the insert. This
method is the least labor intensive because no deletion construction
or generation of random clones is necessary, and template DNA can
be made in the one batch since the template is the same for all sequenc-
ing reactions. However, the delay involved in synthesizing a new oli-
Griffin and Griffin
gonucleotide primer before the next reaction can be performed may
considerably prolong the time taken to sequence a long tract of DNA.
The cost of oligonucleotide synthesis may also be prohibitive.
6. Automation in DNA Sequencing
One of the major advances in sequencing technology in recent years
is the development of automated DNA sequencers. These are based
on the chain termination method and utilize fluorescent rather than
radioactive labels. The fluorescent dyes can be attached to the sequenc-
ing primer, to the dNTPs, or to the terminators, and are incorporated
into the DNA chain during the strand synthesis reaction mediated by
a DNA polymerase (e.g., Klenow fragment of DNA polymerase I,
Sequenase, or Taq DNA polymerase). During the electrophoresis of
the newly generated DNA fragments on a polyacrylamide gel a laser
beam excites the fluorescent dyes. The emitted fluorescence is collec-
ted by detectors and the information analyzed by computer. The data
are automatically converted to nucleotide sequence. Several such
instruments are now commercially available and are becoming increas-
ingly popular (11; Chapters 33-37).
Other aspects of the sequencing procedure that are being automated
include template preparation and purification, and the sequencing
reactions themselves. Robotic workstations are currently being devel-
oped to perform these tasks (Chapter 38).
7. Cycle Sequencing
Cycle sequencing is a new and innovative approach to dideoxy
sequencing. Its advantages over conventional sequencing techniques
are that the reactions are simpler to set up, less template is required,
the quality and purity of template are not as critical, and virtually any
single- or double-stranded DNA can be sequenced (including lambda,
cosmid, plasmid, phagemid, M13, and PCR product). In this method,
a single primer is used to linearly amplify a region of template DNA
using Taq polymerase in the presence of a mixture of dNTPs and a
ddNTP. Either radioactive or fluorescent labels can be used, making
cycle sequencing technology as relevant to automated processes as it
is to manual methods (Chapters 26, 34-36).
As in conventional dideoxy sequencing methods, cycle sequenc-
ing involves the generation of a new DNA strand from a single-stranded
DNA Sequencing
template, synthesis commencing at the site of an annealed primer,
and terminating on the incorporation of a ddNTP. The difference is
that the reaction occurs not just once but 20-30 times under the control
of a thermal cycler (or PCR machine). This results in more and better
sequence data from less template. The process of denaturing a double-
stranded molecule is eliminated, with denaturation occurring auto-
matically in the thermal cycler. The development of cycle sequencing
techniques has made a major contribution to DNA sequencing meth-
odology, improving the reliability and efficiency of DNA sequence
determination and eliminating time-consuming steps.
8. Aim of This Book
The purpose of this book is to provide detailed practical proce-
dures for a number of DNA sequencing techniques. Although proto-
cols for DNA sequencing methods are available elsewhere, there was
a need for a book that comprehensively covered the vanguard tech-
niques now being applied in this rapidly evolving field. Each contri-
bution is written so that a competent scientist who is unfamiliar with
the method can carry out the technique successfully at the first attempt
by simply following the detailed practical procedures that have been
described by each author.
Even the simplest techniques occasionally go wrong, and for this rea-
son a “Notes” section has been included in most chapters. These notes
will indicate any major problems or faults that can occur, their sources,
and how they can be identified and overcome. Since the purpose of this
book is to describe practical procedures and not to go into great depth
regarding theory, a comprehensive reference section is included in
most chapters, enabling the reader to refer to other publications for
more detailed theoretical discussions on the various techniques.
References
1. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing wrth chain-
termmator inhibitors. Proc. Natl. Acad. Sci USA 74, 5463-5467.
2. Maxam, A. M and Gilbert, W (1977) A new method for sequencing DNA
Proc. Natl. Acad. Scl USA 74,560-564.
3. Barrell, B. (1991) DNA sequencmg: present limitations and prospects for the
future. FASEB J 5,40-45
4. Sambrook, J., Frrtsch, E F., and Maniatrs, T. (1989) Molecular Cloning. ,4
Laboratory Manual 2d ed , Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
Griffin and Griffin
5 Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with base-
specific chemical cleavages. Meth. Enzym. 65,499-560.
6. Ambrose, B J. B. and Pless, R. C. (1987) DNA sequencing. Chemical meth-
ods. Meth. Enzymol. 152,522-538.
7. Volckaert, G. (1987) A systematic approach to chemical DNA sequencing by
subcloning in pGV451 and derrved vectors. Meth Enzym. 155,23 l-250
8 Eckert, R. L. (1987) New vectors for rapid sequencing of DNA fragments by
chemical degradation. Gene 51,247-254.
9. Dolz, R. (1993) Fragment assembly programs, in DNA sequencmg: Computer
Analysis ofSequence Data, (Griffin, A. M. and Grrffm, H. G., eds.), Humana
Press, Totowa, NJ. (Ch. 2).
10 Staden, R. (1992) Managing sequencing projects, m DNA sequencing. Com-
puter Analysis of Sequence Data, (Griffin, A. M. and Griffin, H. G., eds.),
Humana Press, Totowa, NJ. (Ch 17).
11 Hunkapiller, T , Karser, R J., Kopp, B F , and Hood, L. (1991) Large-scale
and automated DNA sequence determination. Science 254,59-67
&IAE’TER 2
Ml3 Cloning Khicles
Their Contribution to DNA Sequencing
Joachim Messing
1. Introduction
For studies in molecular biology, DNA purification has been essen-
tial, in particular for DNA sequencing, probing, and mutagenesis.
The amplification of DNA in E. coli by cloning vehicles derived from
M13mp or pUC made expensive physical separation techniques like
ultracentrifugation unnecessary.A lthough today the polymerase chain
reaction is a valuable alternative for the amplification of small DNA
pieces (I), it cannot substitute for the construction of libraries of DNA
fragments. Therefore, E. coli has served not only as a vehicle to
amplify DNA, but also to separate many DNA molecules of similar
length and the two DNA strands simultaneously. For this purpose, a
bacteriophage like M 13 can be used. The various viral cis- and truns-
acting functions are critical not only for strand separation, but also to
separate the single-stranded DNA from the E. coli cell by an active
transport mechanism through the intact cell wall.
Although it may have been somewhat surprising to some how many
changes in its DNA sequence the phage tolerated, manipulations of
this amplification and transport system have been extended today even
to the viral coat proteins for the production of epitope libraries (2).
Much of the work is now more than a decade old, but experience has
confirmed the usefulness of some simple biological paradigms. Tech-
From Methods m Molecular Biology, Vol 23’ DNA Sequencmg Protocols
Edlted by. H and A Gnffm Copyright 01993 Humana Press Inc , Totowa, NJ
9
10 Messing
niques that were new and limiting fifteen years ago included auto-
mated oligonucleotide synthesis and the use of thermostable enzymes,
which add a critical dimension to molecular biology today. Neither
necessarily replaces the previous techniques, but they create greater
flexibility, enormously accelerate scientific investigations, and even
make certain analyses possible for the first time.
However, DNA sequencing of larger contigs (several overlapping
sequences that can be linked) have benefited from economizing on
the synthesis of new oligonucleotides (3,4). Even in the absence of
automated oligonucleotide synthesis in the earlier years, the concept
of a universal primer could be developed by alternative techniques.
2. Development of DNA Sequencing Techniques:
A Discussion
In 1974, at one of the first meetings on the use of restriction endo-
nucleases in molecular biology some of these ideas became clear,
Work on the chemical synthesis of a tRNA gene was presented, and
the initial work on sequencing phage $X174 using restriction frag-
ments as primers for the plus-minus method was discussed. At that
time, oligonucieotide synthesis required a major effort and could not
easily be generally applied. Restriction fragments offered an alterna-
tive. They could be used as primers for DNA synthesis in vitro and
for marker-rescue experiments to link genetic and physical maps of
viruses like SV40, both forerunners for DNA sequencing and site-
directed mutagenesis.
There were several reasons to use @X 174 as the first model in devel-
oping DNA-sequencing techniques and determining the sequence of
an entire autonomous genome. First, it was one of the smallest DNA
viruses; it is even smaller than M13. Second, the mature virus con-
sists of single-stranded DNA, eliminating the need to separate the
two strands of DNA for template preparation; this is even more criti-
cal if one wishes to use double-stranded restriction fragments as prim-
ers. Third, a restriction map was superimposed on the genetic map by
marker-rescue experiments (5). The latter feature still serves today as
a precondition for other genomes. Restriction sites were critical as
signposts along the thousands of nucleotides and provided the means
to dissect the double-stranded replicative form or RF of @X174 in
small, but ordered pieces that permitted the DNA sequencing effort
