Rob Beynon UMIST, Manchester, UK Chris Howe Department of Biochemistry, University of Cambridge
Rob Beynon UMIST, Manchester, UK Chris Howe Department of Biochemistry, University of Cambridge
Rob Beynon UMIST, Manchester, UK Chris Howe Department of Biochemistry, University of Cambridge
M.J.McPherson
School of Biochemistry and Molecular Biology,
University of Leeds, Leeds, UK and
S.G.Møller
Laboratory of Plant Molecular Biology,
Rockefeller University, New York, USA
© BIOS Scientific Publishers 2000
First published 2000
This edition published in the Taylor & Francis e-Library, 2005.
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any electronic, mechanical, or other means, now known or hereafter invented, including
photocopying and recording, or in any information storage or retrieval system, without permission in
writing from the publishers.
A CIP catalogue record for this book is available from the British Library.
Abbreviations x
Preface xii
1. Introduction 252
2. Why map genomes? 253
3. Single-strand conformation polymorphism analysis (SSCP) 255
4. Ligase chain reaction 259
5. Amplification refractory mutation system (ARMS) 260
6. Single-tube Tm-shift genotyping 262
7. Cleaved amplified polymorphic sequence analysis (CAPS) 265
8. Random amplified polymorphic DNA (RAPD) 265
9. Amplified fragment length polymorphisms (AFLPs) 267
10. Multiplex PCR analysis of Alu polymorphisms 267
11. Variable number tandem repeats in identity testing 271
12. Minisatellite repeat analysis 274
13. Microsatellites 276
14. Sensitive PCR for environmental and diagnostic applications277
15. Screening transgenics 279
Appendix 283
Web addresses of suppliers of PCR related products 283
Index 285
Abbreviations
8-MOP 8-methoxypsoralen
AFLP amplified length polymorphism
AMV avian myeloblastoma virus
AP alkaline phosphatase
AP-PCR arbitrarily primed PCR
ARMS amplification refractory mutation system
ASA allele specific amplification
ASP allele-specific PCR
BCIP 5-bromo, 4-chloro, 3-indolyl phosphate
CAPS cleaved amplified polymorphic sequence analysis
CCD charge coupled device
cDNA complementary DNA
DIG digoxigenin
DIG-dUTP digoxigenin-11-2′
-deoxyuridine-5′-triphosphate
ELISA enzyme linked immunosorbent assay
EST expressed sequence tag
FAM 6 carboxyfluorescein
FDD fluorescent differential display
FRET fluorescence energy transfer
FS fluorescent sequencing
GAWTS gene amplification with transcript sequencing
GM genetically modified
HEX ,4′,5′,7′-hexachloro-6-carboxyfluorescein
4,7,2′
HRP horseradish peroxidase
IPCR inverse polymerase chain reaction
LCR ligase chain reaction
LIC ligation-independent cloning
xi
The polymerase chain reaction (PCR) has become an integral tool for the
molecular biologist. It represents a workhorse in the molecular biology
laboratory whilst also proving to be a highly versatile process which is readily
adaptable, by the imaginative researcher, for a wide variety of new procedures
and applications. This book is designed to provide an introduction for those new
to PCR and we hope that it will also prove to be of value to those more
experienced with the technology The intention has been to provide both an
understanding of PCR from a theoretical perspective and guidelines about how to
begin using it experimentally It is also intended to provide an introduction to the
range of applications allowing the reader to understand their specific
requirements in greater detail. However, it is important to stress that there has
been no attempt to describe every application or variation of PCR, a task that
would require a significantly larger volume.
Initially, the book provides an introduction to the concept of PCR before
considering the molecular basis of the process and a basic protocol. This is
followed by a consideration of practical aspects of PCR including the various
essential reagents and equipment, such as thermostable DNA polymerases and
thermal cycling instruments central to the methodology. Aspects of this section
provide a technological “snapshot” of available products to give the reader some
view of the wide product range available for PCR experimentation. However, the
speed with which new products continue to emerge means that you should
explore manufacturer’s catalogs and web sites to determine the latest and most
appropriate products for your specific needs. The book then proceeds to further
practical issues including optimization of PCR, avoiding contamination and
product analysis, including confirming correct product amplification, direct
sequencing and real time monitoring systems. There follows a discussion of the
purification and cloning of PCR products. The remainder of the book describes
some applications of PCR, including mutagenesis, analyzing gene expression
profiles, cloning expressed and genomic genes, and finally some aspects of
genome analysis.
We are grateful to Clive Newton and Alex Graham for providing access to the
text of their excellent book, PCR, from the Introduction to Biotechniques series
xiii
(BIOS Scientific Publishers Ltd). This volume provided a useful basis for
aspects of the current book and provided a starting point for a few sections.
The various experimental sections of this book have been written with safety
in mind, but the user should ensure that they take all necessary precautions to
ensure safe practice of the protocols. The authors accept no liability whatsoever
for any injury or loss resulting from use of this book. It should also be noted that
purchase of this book does not confer any license to perform PCR covered by
any patents related to the PCR process.
We are extremely grateful to those who have kindly provided illustrations used
in this book, including Dr Anil Neelam (Figures 6.7, 9.1, 9.2, 9.3, 10.10) and
David Harrison (Figure 7.17) of the University of Leeds, Dr Luis Lopez-Molina
(Figure 4.3) of Rockefeller University, CLONTECH Laboratories, Inc.
(Figure 8.4; Figure 8.5), Sarah Batt and Dr Bambos Charalambous
(Figure 9.15) of the Royal Free School of Medicine, Dr S.Germer and Dr
R.Higuchi (Figure 10.6) of Roche Molecular Systems (RMS) and Dr R J Herrera
(Figures 10.7, 10.8) of Florida International University. We also thank Dr Donal
McGrogan of Memorial Sloan-Kettering Cancer Center for discussions on
genome mapping techniques. We thank the staff at BIOS for their patience in
dealing with academic authors and for their speed and efficiency during the
publication process.
Chapter 1
An introduction to PCR
1.
Introduction: PCR, a ‘DNA photocopier’
Does it really work? It is so simple! Why did I not think of it? These thoughts
were probably typical of most molecular biologists on reading early reports of
the polymerase chain reaction or PCR as it is more commonly called. PCR uses a
few basic components, ones that we use in the laboratory every day to make
large numbers of copies of a specific DNA fragment in a test-tube. PCR has been
called a ‘DNA photocopier’. Although apparently simple, PCR is a complicated
process with many reactants. Some are present at very low initial concentrations
(template) which increase dramatically as the reaction proceeds, others are at
concentrations that hardly change during the reaction (dNTPs, primers). There
are significant changes in temperature and pH and therefore dramatic
fluctuations in the dynamics of a range of molecular interactions. So PCR is
really a very complex process, but one with tremendous power and versatility for
DNA manipulation and analysis.
In the short time since its invention by Kary Mullis, PCR has revolutionized
our approach to molecular biology. The impact of PCR on biological and medical
research has been like a supercharger in an engine, dramatically speeding the rate
of progress of the study of genes and genomes. We can now isolate essentially
any gene from any organism using PCR, and it has become a cornerstone of
genome sequencing projects. Having isolated a gene we can tailor its sequence to
allow straightforward cloning or mutagenesis and we can establish diagnostic
tests to detect mutant forms of a gene. Since the first reports of a functional PCR
procedure there has been almost an exponential increase, not only in the amount
of DNA synthesized by PCR, but also in the number of scientific papers
describing new applications or new methods (Figure 1.1). Several books and a
new journal have been published and many commercial products and kits have
been launched for PCR research and for PCR-based diagnostics, and some of
these will be discussed in later chapters.
2 PCR
Figure 1.1
Approximate number of papers published annually that quote use of the polymerase chain
reaction in their titles, keywords or abstracts. Taken from ISI Citation Database at http://
www.wos.mimas.ac.uk.
2.
History of PCR
As long ago as 1971, Khorana and colleagues described an approach for
replicating a region of duplex DNA using two DNA synthesis primers designed
so that their 3′-ends pointed towards each other (Kleppe et al., 1971). However,
the concept of using such an approach repeatedly in an amplification format was
not conceived for another 12 years. ‘Sometimes a good idea comes to you when
you are not looking for it.’ With these words Kary Mullis, the inventor of PCR,
started an account in Scientific American of how, during a night drive through
the mountains of Northern California in Spring 1983, he had a revelation that led
him to develop PCR. The practical aspects of the PCR process were then
developed at Cetus Corporation, the company for which Mullis worked, and
became a major part of their business, before they finally sold the rights to PCR
in 1991 for $300m.
Since the potential of PCR was realized, it has become rather entangled in
commercialism owing to the large amounts of money to be made from licensing
the technology The PCR is covered by patents, granted to Hoffman La-Roche
and Roche Molecular Systems, and these are vigorously enforced to prevent
unlicensed use of the method. Relevant patents include US Patent numbers 4,683,
202, 4,683,195, 4,800,159 and 4,965,188 covering the PCR process, and 4,889,
818, 5,075,216 and 5,079,352 covering Taq and AmpliTaq® DNA polymerases.
Any organization, and this includes academic and public organizations, is
required to enter into licensing agreements for access to PCR technology, which
includes both reagents and thermal cycler instruments. Academic laboratories are
granted a license to use the PCR through the purchase of the GeneAmp reagents
AN INTRODUCTION TO PCR 3
2.1
Key milestones in the development of PCR
3.
PCR involves DNA replication
PCR copies DNA in the test tube and uses the basic elements of the natural DNA
replication processes. In a living cell a highly complex process involving many
different proteins replicates the complete genome. In simplistic terms, the DNA
is unwound and each strand of the parent molecule is used as a template to produce
a complementary ‘daughter’ strand. This copying relies on the ability of
nucleotides to base pair according to the well-known Watson and Crick rules: A
always pairs with T and G always pairs with C. The template strand therefore
specifies the base sequence of the new DNA strand. A large number of proteins
and other molecules, such as RNA primers, are required to ensure that the
process of DNA replication occurs efficiently with few mistakes (high fidelity)
and in a tightly regulated manner.
PCR uses only the basic components of this complex replication machinery to
replicate short fragments of DNA in a test tube. In a simple buffer system a
region of a template DNA molecule is copied by a DNA polymerase that uses
deoxynucleotides as building blocks of the new strands. Sequence-specific
oligonucleotide primers that bind to the template according to normal base-
pairing rules (Figure 1.2) define the region of template to be copied. The strands
of the template are separated by heat, which causes the hydrogen bonds between
the base pairs of the DNA strands to break, a process known as denaturation. The
oligonucleotide primers then find their complementary sequences on the
templates (annealing) and DNA polymerase then begins to add deoxynucleotides
AN INTRODUCTION TO PCR 5
Figure 1.2
Oligonudeotides base-pairing according to Watson-Crick base-pairing rules with their
corresponding template DNA strands. The 3′-ends of the oligonudeotides will be extended
by DNA polymerase.
to the 3′-OH group of the primers, producing new duplex molecules
(Figure 1.2). At the next heating step, these new double-stranded molecules are
once again denatured and each single strand (both the original templates and newly
synthesized strands) provides a primer binding site and acts as a template for
further DNA synthesis. In this step, as discussed in Chapter 2, the first DNA
strands of the defined length of the expected PCR product are formed due to
priming of the second primer on the template generated in the first cycle by
priming from the first primer. There is then an exponential increase in the number
of copies of the ‘target’ DNA sequence; the number of strands of the target
sequence will be doubled at each PCR cycle (see Figure 2.1 and Table 2.1).
This means that, at 100% efficiency, each template present at the start of the
reaction would give rise to 106 new strands after only 20 cycles of PCR. The
process is not 100% efficient, and it is usually necessary to carry out more
reaction cycles, often 25–40 depending upon the concentration of the starting
template and the application. However, very low numbers of molecules present at
the start of the PCR can be amplified into a large amount of DNA, often a
microgram or more, which is plenty for detailed analysis in a range of
applications. Of course it also means that if you happen to contaminate your
reaction with a few molecules of DNA you may get a false result. We will deal with
contamination problems in Chapter 4.
4.
PCR is controlled by heating and cooling
PCR relies on the use of different temperatures for the three steps of the reaction
—denaturation, annealing and extension. A high temperature, usually 94–95°C,
is used to separate the strands of the DNA template. The temperature is then
lowered to allow the primers to base pair to their complementary sequences on
the template strands and this temperature varies depending on the primers (see
Chapter 3). Finally, for efficient DNA synthesis, the temperature is adjusted to
be optimal for the DNA polymerase activity (see Chapter 3). To amplify the
6 PCR
Figure 1.3
Primer extension by a DNA polymerase. The primer anneals to a complementary
sequence on the template strand and the DNA polymerase uses the template sequence to
extend the primer by incorporation of the correct deoxynucleotide (dNTP).
target DNA it is necessary to cycle through these temperatures several times (25–
40 depending on the application). This is accomplished using a thermal cycler, a
programmable instrument that can rapidly alter temperature and hold the tubes at
the desired temperature for a predetermined time. This automation is one of the
important advances that made PCR widely accessible and is covered in more
detail in Chapter 3. Before thermal cyclers, PCR was performed using three water
baths at set temperatures where the reaction tubes were moved manually between
the baths.
The other major advance was the replacement of Klenow polymerase with
thermostable DNA polymerases such as Taq DNA polymerase, which are not
inactivated at the high denaturation temperatures used during PCR. The ability to
carry out the steps of the extension at high temperatures has the added benefit
that it enhances the specificity of the reaction (Chapter 4). At lower temperatures
such as 37°C, where Klenow works best, primers can bind to other sequences
because mismatches between the two strands can be tolerated, thereby decreasing
the specificity. There have now been numerous developments that have
simplified the PCR process, allowing it to be used in all molecular biology
laboratories as a routine procedure.
AN INTRODUCTION TO PCR 7
5.
Does PCR replace gene cloning?
In some cases PCR is an alternative to gene cloning, but in other cases it is not.
In gene cloning, a fragment of DNA is joined, by ligation, to a cloning vector which
is able to replicate within a host cell such as the bacterium Escherichia coli. As
the bacterium grows, the new recombinant DNA molecule is copied by DNA
replication and, as the cell divides, the number of cells carrying the recombinant
molecule increases. Finally when there are enough cells you can isolate the
recombinant DNA molecules to provide sufficient DNA for analysis or further
manipulation of the cloned DNA fragment. This type of cloning experiment
takes about 3 days.
PCR also amplifies your target DNA fragment so that you have enough to
analyze or manipulate, but in this case the DNA replication occurs in a test tube
and usually takes no more than 3–4 h. In many cases, for example in diagnostic
tests for cancers or genetic diseases including antenatal screening, the PCR has
greatly simplified genetic analysis because the PCR product can be generated
and analyzed within a day However, you should not think of PCR as a universal
replacement for gene cloning methods. You should think of them as
complementary molecular tools. In the case of routine genetic testing, it clearly
is a replacement, but for studying new genes and genetic diseases it is not. In
research investigations you can use PCR to amplify a target DNA fragment
which you then clone for further analysis. So PCR and gene cloning go hand-in-
hand, and you need to think carefully about what experiments you want to do
before you decide on an appropriate PCR strategy, as we will describe in
Chapter 6.
6.
PCR applications
The speed and simplicity of PCR technology accompanied by an increased range
of high-quality products has led to a more rational approach to PCR
experimentation. We understand better the molecular processes underlying PCR
(see Chapter 2) so that it is seen less as a ‘witches-brew’. In particular it is
important to highlight good practices that increase confidence in results by
reducing the likelihood of artifactual results. The importance of good PCR
technique, particularly with regard to proper controls and the prevention of
contamination (Chapter 4) cannot be overemphasized. Remember, if you work in
a research laboratory a wrong result may be inconvenient, leading to a waste of
time, effort and money and so should be avoided. However, if you work in a
diagnostic laboratory, a wrong result could mean the difference between life and
death. It is a good idea to start with the highest standards and expectations so
that you can be confident in your results no matter where you work.
8 PCR
PCR has now been adapted to serve a variety of applications and some of
these will be described in detail in this book (Chapters 5–10). PCR has
revolutionized our approach to basic scientific and medical research, to medical,
forensic and environmental testing. It provides an extremely flexible tool for the
research scientist, and every molecular biology research laboratory now uses
PCR routinely, often adapting and tailoring the basic procedures to meet their
own special needs. At the other end of the spectrum PCR is also a routine tool
for repetitive DNA analyses such as diagnosis of certain genetic diseases within
clinical screening laboratories, where speed and accuracy are important factors.
A useful introduction to PCR is available at Paul Rabinow’s website http://
sunsite.berkeley.edu/pcr and a simple animation of the first cycles of PCR is
available at http://wsrv.clas.virginia.edu/~rjh9u/pcranim.html
Further reading
Mullis, K.B. (1990) The unusual origins of the polymerase chain reaction. Sci. Am. 262,
56–65.
White, T.J. (1996) The future of PCR technology: diversification of technologies and
applications. Trends Biotechnol. 14, 478–483.
References
Kleppe, K., Ohtsuka, E., Kleppe, R., Molineux, R. and Khorana, H.G. (1971) Studies
on polynucleotides. XCVI. Repair replication of short synthetic DNA’s as catalysed
by DNA polymerases. J. Mol. Biol. 56, 341–346.
Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and
Arnheim, N. (1985) Enzymatic amplification of beta-globin genomic sequences and
restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S., Higuchi, R., Horn, G.T., Mullis,
K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with
a thermostable DNA polymerase. Science 239, 487–491.
References
Protocol
Notes
Poinar, H.N., Cano, R.J. and Poinar, G.O. (1993) DNA from
an extinct plant. Nature 363, 677.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1981)
Molecular Cloning: a Laboratory manual (2nd edn). Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Protocol
Protocol
Protocol
Protocol
5. Add 0.1 vol 3 M NaOAc (pH 5.2) and mix, then add 2.5 vol
ice-cold ethanol and allow the DNA to precipitate for 1 h
at room temperature.
Kaneoka, H., Lee, D.R., Hsu, K.C., Sharp, G.C. and Hoffman,
R.W. (1991) Solidphase direct DNA sequencing of
allele-specific polymerase chain reaction-amplified HLA-DR
genes. BioTechniques 10, 30.
Protocol
Protocol
Protocol
12. Aspirate off the ethanol, gently tap the mouth of the
tube onto some tissue to remove most of the residual
ethanol and dry briefly (up to 5 min) in a centrifugal
evaporator to remove traces of ethanol. Do not overdry the
DNA as this makes redissolving difficult.
Figure 6.7
Protocol
Protocol
appropriate cultures.
1. Pipette a 20 μ l aliquot of a PCR reactants premix into
a PCR tube.
Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease,
L.R. (1989) Engineering hybrid genes without the use of
restriction enzymes: gene splicing by overlap extension.
Gene 77, 61–68.
Zhao, H., Giver, L., Shao, Z., Affholter, J.A. and Arnold,
F.H. (1998) Molecular evolution by staggered extension
process (StEP) in vitro recombination. Nature Biotechnology
16, 258–261. Protocol 7.1 Splicing by overlap extension
(SOEing) Equipment 0.5 or 0.2 ml PCR tubes Microcentrifuge
Thermal cycler Gel electrophoresis tank
Protocol
′ 5 mM each dNTP;
′ 5 mM ATP;
′ 100 mM DTT;
Protocol
′ 2 µl 10×polymerase buffer;
′ 2 µl 2 mM dNTPs;
Note
′ 5 μ g template DNA;
′ 95°C, 30s;
′ 50°C, 30s;
′ 72°C, 1 min;
′ total volume 50 μ l.
′ 10×buffer, 4 µl;
′ dNTPs, 4 µl;
′ polymerase, 5 units;
′ total volume, 40 μ l.
′ dNTPs, 2.5 μ l;
′ Polymerase, 5 units;
′ Total volume, 25 μ l.
′ 92°C, 30s;
′ 55°C, 30s;
′ 72°C, 1 min;
′ polymerase, 5 units;
′ total volume, 15 μ l;
′ dNTPs, 2 µl;
′ polymerase, 2.5 units;
′ dNTPs, 2 µl;
Protocol
′ 1 mM of each dNTP
′ Water up to 49 μ l.
Protocol Part B—preparing the gel mix for a 0.4 mm×40 cm×20
cm gel
Most things can go wrong when you are pouring the gel: the
gel may leak, set
before it is completely poured or you may introduce
air-bubbles in your gel. Try
to relax!
4. Pour the gel slowly and continuously down the right side
of the sandwich so that it starts to fill at the corner
that is on the bench.As the sandwich begins to fill you can
gradually lower your left hand so that the bottom edge is
resting completely on the bench. It is important to
maintain a continuous flow of acrylamide to prevent the
introduction of air bubbles.
8. Allow the gel to cool, then remove the tape and lay the
gel with the silanized (notched) plate uppermost. Remove
the spacers if possible by gently pulling them from the
sandwich.
11. Dry the transferred gel in a gel dryer under vacuum and
expose to X-ray film at −70°C for varying length of time
depending on the intensity of the signal. Protocol 8.3
Recovery of DNA from the dried gel Equipment Razor blades
Disposable pestle Microcentrifuge
Protocol
2. Cut out the dried gel slice using a sharp razor blade.
simple:
Lee, C.C., Wu, X.W., Gibbs, R.A., Cook, R.G., Muzny, D.M.
and Caskey, C.T. (1988) Generation of cDNA probes directed
by amino acid sequence: cloning of urate oxidase. Science
239, 1288–1291.
′ 1 µl of 10 mM dNTP;
′ 10 μ l 5×tailing buffer;
′ 2.5 µl of 2 mM dCTP;
′ Water up to 49 µl.
′ 5 µl 10×PCR buffer;
′ 2 µl 10 mM dNTP;
′ 20 μ M of GSP2;
′ 20 μ M of poly-G primer;
′ 5 μ l of tailed cDNA;
′ water up to 49.5 µl
Protocol
′ 2 µl 10 mM dNTP;
′ 5 μ l of 10×PCR buffer;
′ water up to 50 µl.
′ 2 µl 10 mM dNTP;
′ 5 µl of 10×PCR buffer;
′ water up to 50 μ l.
Figure 10.10
(University of Leeds).
Sabat, G., Rose, P., Hickey, W.J. and Harkin, J.M. (2000)
Selective and sensitive method for PCR amplification of
Escherichia coli 16S rDNA genes in soil. Appl. Environ.
Microbiol. 66, 844–849.