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PCR
Second Edition

Michael J. McPherson
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences,
University of Leeds, Leeds, UK
and

Simon Geir Møller

Faculty of Science and Technology
Department of Mathematics and Natural Sciences
University of Stavanger
N-4036 Stavanger
Norway
Published by:
Taylor & Francis Group

In US: 270 Madison Avenue

New York, N Y 10016
In UK: 4 Park Square, Milton Park
Abingdon, OX14 4RN
This edition published in the Taylor & Francis e-Library, 2006.
“To purchase your own copy of this or any of Taylor & Francis or Routledge’s
collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.”

© 2006 by Taylor & Francis Group

First published 2000; Second edition published 2006

ISBN 0-203-00267-9 Master e-book ISBN

ISBN: 0-4153-5547-8 (Print edition)

This book contains information obtained from authentic and highly regarded sources. Reprinted material is
quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable
efforts have been made to publish reliable data and information, but the author and the publisher cannot
assume responsibility for the validity of all materials or for the consequences of their use.

All rights reserved. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form
by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying,
microfilming, and recording, or in any information storage or retrieval system, without written permission
from the publishers.

A catalog record for this book is available from the British Library.
____________________________________________________________________
Library of Congress Cataloging-in-Publication data has been applied for.

Editor: Elizabeth Owen

Editorial Assistant: Kirsty Lyons
Production Editor: Karin Henderson
Typeset by: Phoenix Photosetting, Chatham, Kent,UK
Printed by: MPG BOOKS Limited, Bodmin, Cornwall, UK

Taylor & Francis Group

is the Academic Division of Informa UK Limited Visit our web site at http://www.garlandscience.com
Contents

Abbreviations ix
Preface xi

Chapter 1 An Introduction to PCR 1

1.1 Introduction: PCR, a ‘DNA photocopier’ 1
1.2 PCR involves DNA synthesis 1
1.3 PCR is controlled by heating and cooling 3
1.4 PCR applications and gene cloning 5
1.5 History of PCR 6

Chapter 2 Understanding PCR 9

2.1 How does PCR work? 9
2.2 PCR: a molecular perspective 11
2.3 The kinetics of PCR 15
2.4 Getting started 18
2.5 Post-PCR analysis 18
Protocol 2.1: Basic PCR 20

Chapter 3 Reagents and Instrumentation 23

3.1 Technical advances in PCR 23
3.2 Reagents 23
3.3 PCR buffers 23
3.4 Nucleotides 25
3.5 Modified nucleotides 25
3.6 PCR premixes 26
3.7 Oligonucleotide primers 26
3.8 DNA polymerases for PCR 36
3.9 Early PCR experiments 37
3.10 Thermostable DNA polymerases 37
3.11 Properties of Taq DNA polymerase 37
3.12 Thermostable proofreading DNA polymerases 43
3.13 Tth DNA polymerase has reverse transcriptase activity 46
3.14 Red and green polymerases and reagents 47
3.15 Polymerase mixtures: high-fidelity, long-range and RT-PCRs 48
3.16 Nucleic acid templates 51
3.17 Mineral oil 54
3.18 Plasticware and disposables 54
3.19 Automation of PCR and thermal cyclers 55
Protocol 3.1: Phosphorylation of the 5′-end of an oligonucleotide 63
vi Contents

Chapter 4 Optimization of PCR 65

4.1 Introduction 65
4.2 Improving specificity of PCR 65
4.3 Template DNA preparation and inhibitors of PCR 75
4.4 Nested PCR improves PCR sensitivity 76
4.5 Contamination problems 76
4.6 Preventing contamination 80
4.7 Troubleshooting guide 82

Chapter 5 Analysis, Sequencing and In Vitro Expression of

PCR Products 87
5.1 Introduction 87
5.2 Analysis of PCR products 87
5.3 Verification of initial amplification product 89
5.4 Direct DNA sequencing of PCR products 93
5.5 Direct labeling of PCR products and homogenous assays 101
5.6 In vitro expression of PCR product 103
Protocol 5.1: Cycle sequencing – Applied Biosystems Big Dye
terminators 108

Chapter 6 Purification and Cloning of PCR Products 111

6.1 Introduction 111
6.2 Purification of PCR products 111
6.3 Introduction to cloning of PCR products 115
6.4 Approaches to cloning PCR products 117
6.5 Confirmation of cloned PCR fragments 131
Protocol 6.1: Blunt-end polishing of PCR fragments 134
Protocol 6.2: PCR screening of bacterial colonies or cultures 135

Chapter 7 PCR Mutagenesis 137

7.1 Introduction 137
7.2 Inverse PCR mutagenesis 138
7.3 Unique sites elimination 144
7.4 Splicing by overlap extension (SOEing) 144
7.5 Point mutations 150
7.6 Deletions and insertions 151
7.7 Deletion mutagenesis 151
7.8 Insertion mutagenesis 151
7.9 Random mutagenesis 157
7.10 PCR misincorporation procedures 159
7.11 Recombination strategies 160
7.12 RACHITT 166
7.13 Gene synthesis 166
Protocol 7.1: Inverse PCR mutagenesis 171
Protocol 7.2: Quikchange mutagenesis of plasmid DNA 173
Protocol 7.3: Splicing by overlap extension (SOEing) 175
Protocol 7.4: ‘Sticky-feet’ mutagenesis 177
 Contents vii

Protocol 7.5: DNA shuffling 179

Protocol 7.6: Gene synthesis 182

Chapter 8 Analysis of Gene Expression 185

8.1 Introduction 185
8.2 Reverse transcriptase PCR (RT-PCR) 185
8.3 Semi-quantitative and quantitative RT-PCR 189
8.4 One-tube RT-PCR 194
8.5 Differential display 194
8.6 PCR in a cell: in situ RT-PCR 198
8.7 Microarrays 204
8.8 RNA interference (RNAi) 205
Protocol 8.1: Reverse transcriptase reaction 208

Chapter 9 Real-Time RT-PCR 209

9.1 Introduction 209
9.2 Basic principles of real-time RT-PCR 209
9.3 Detection methods 212
9.4 General guidelines for probe and primer design 221
9.5 Instruments and quantification of results 222
9.6 Normalization and control selection 225
9.7 A typical real-time RT-PCR experiment using SYBR® Green I 225
9.8 Common real-time RT-PCR pitfalls 228
9.9 Applications of real-time RT-PCR 229

Chapter 10 Cloning Genes by PCR 233

A Cloning genes of known DNA sequence 233
10.1 Using PCR to clone expressed genes 233
10.2 Express sequence tags (EST) as cloning tools 237
10.3 Rapid amplification of cDNA ends (RACE) 238
B Isolation of unknown DNA sequences 240
10.4 Inverse polymerase chain reaction (IPCR) 240
10.5 Multiplex restriction site PCR (mrPCR) 243
10.6 Vectorette and splinkerette PCR 244
10.7 Degenerate primers based on peptide sequence 248
Protocol 10.1: 5′-RACE 253
Protocol 10.2: Inverse PCR from plant genomic DNA 255

Chapter 11 Genome Analysis 257

11.1 Introduction 257
11.2 Why map genomes? 258
11.3 Single-strand conformation polymorphism analysis (SSCP) 259
11.4 Denaturing-high-performance liquid chromatography
(DHPLC) 263
11.5 Ligase chain reaction (LCR) 264
11.6 Amplification refractory mutation system (ARMS) 264
viii Contents

11.7 Cleaved amplified polymorphic sequence analysis (CAPS) 267

11.8 SNP genotyping using DOP-PCR 268
11.9 Random amplified polymorphic DNA (RAPD) PCR 269
11.10 Amplified fragment length polymorphisms (AFLPs) 270
11.11 Multiplex PCR analysis of Alu polymorphisms 270
11.12 Variable number tandem repeats in identity testing 271
11.13 Minisatellite repeat analysis 274
11.14 Microsatellites 276
11.15 Sensitive PCR for environmental and diagnostic applications 277
11.16 Screening transgenics 278
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Abbreviations

8-MOP 8-methoxypsoralen FRET fluorescence resonance

8-oxo-dGTP 8-oxo-2′deoxyguanosine energy transfer
AFLP amplified length FS fluorescent sequencing
polymorphism GAPDH glyceraldehyde-3-phosphate
AMV avian myeloblastoma virus dehydrogenase
AP alkaline phosphatase GAWTS gene amplification with
AP-PCR arbitrarily primed PCR transcript sequencing
ARMS amplification refractory GM genetically modified
mutation system HEX 4,7,2′,4′,5′,7′-hexachloro-6-
ASA allele specific amplification carboxyfluorescein
ASP allele-specific PCR HRP horseradish peroxidase
BAC bacterial artificial IPCR inverse polymerase chain
chromosome reaction
BCIP 5-bromo, 4-chloro, 3- LCR ligase chain reaction
indolyl phosphate LIC ligation-independent
CAPS cleaved amplified cloning
polymorphic sequence M-MLV Moloney murine leukemia
analysis virus
CcdB control of cell death MPSV mutations, polymorphisms
Ct threshold cycle and sequence variants
CCD charge coupled device mrPCR multiplex restriction site
cDNA complementary DNA PCR
CT comparative threshold MVR minisatellite variant repeat
DHPLC denaturing-high- NBT nitro blue tetrazolium
performance liquid NF nonfluorescent
chromatography nt nucleotides
DIG digoxigenin ORFs open reading frames
DIG-dUTP digoxigenin-11-2′- PAGE polyacrylamide gel
deoxyuridine-5′- electrophoresis
triphosphate PASA PCR amplification of
DOP-PCR degenerate oligonucleotide specific alleles
primed-PCR PBS phosphate buffered saline
dPTP 6-(2-deoxy-β-D- PCR polymerase chain reaction
ribofuranosyl)-3,4-dihydro- PCR-VNTRs PCR highly polymorphic
8H-pyrimido-[4,5-C][1,2] variable number tandem
oxazin-7-one repeats
ELISA enzyme linked PEETA Primer extension,
immunosorbent assay Electrophoresis, Elution,
EST expressed sequence tag Tailing, Amplification
FAM 6-carboxyfluorescein PMBC peripheral blood
FDD fluorescent differential mononuclear cells
display PMT photomultiplier tube
x Abbreviations

PNA peptide nucleic acid StEP staggered extension

PORA NADPH: process
protochlorophyllide STR short tandem repeats
oxidoreductase TAIL-PCR thermal asymmetric
RACE rapid amplification of cDNA interlaced PCR
ends TAMRA 6-carboxytetramethyl-
RACHITT random chimeragenesis on rhodamine
transient templates TBR tris (2,2′-bipyridine)
RAPD random amplified ruthenium (II) chelate
polymorphic DNA TCA trichloroacetic acid
RAWIT RNA amplification with in TdT terminal deoxynucleotidyl-
vitro translation transferase
RAWTS RNA amplification with TEMED N,N,N′,N′-
transcript sequencing tetramethylenediamine
RFLP restriction fragment length TET 4,7,2′,7-tetrachloro-6-
polymorphism carboxy fluorescein
RISC RNA-induced silencing TK thymidine kinase
complex Tm melting temperature
RNAi RNA interference TNF tumor necrosis factor
RT reverse transcriptase TOPO ligation topoisomerase-mediated
SDS sodium dodecyl sulfate ligation
siRNAs small interfering RNAs Tp optimized annealing
SNPs single nucleotide temperature
polymorphisms UNG uracil N-glycosylase
SOEing splicing by overlap USE unique site elimination
extension VNTR variable number tandem
SPA scintillation proximity assay repeats
SSCP single strand conformation YAC yeast artificial
polymorphism analysis chromosome
Preface

The concept underlying this book has not changed from the first edition; it is to provide
an introductory text that is hopefully useful to undergraduate students, graduate students
and other scientists who want to understand and use PCR for experimental purposes.
Although applications of PCR are provided these do not represent a comprehensive
catalogue of all possible PCR applications, but serve to indicate the types of application
possible. The main purpose of this new edition of PCR, as for the first edition, is to
provide information on the fundamental principles of the reactions occurring in a PCR
tube. Understanding these basic features is essential to fully capitalize upon and adapt the
power of PCR for a specific application. This means that the structure of the book remains
similar to that of the first edition. The first six Chapters discuss the fundamental aspects
of performing PCR and of analyzing and cloning the products. All these Chapters have
been updated and additional aspects added where appropriate. In some Sections there is
discussion of particular enzymes or instruments. However, clearly suppliers are continually
changing their formulations or designs and so these are provided only to indicate the
different types. We recommend checking manufacturers’ literature for new and improved
systems, particularly when it comes to investing in the purchase of a new PCR instru-
ment. In terms of the applications, a new Chapter has been written on real-time PCR,
which represents a very sensitive and reliable method for providing information about the
relative concentrations of starting template molecules, such as mRNA or genomic genes.
The remaining Chapters have been updated and Protocols have been rationalized to retain
those that are likely to be the most useful. We have also removed the list of web addresses
of various reagent suppliers. Such lists can quickly become outdated and it is simpler for
the reader to identify the up to date website from a web search engine. We hope that this
book will provide the basic information required to get scientists started with PCR experi-
ments either to use it simply as a routine tool, or as a starting point for developing new
and innovative processes.
We thank those who kindly provided figures to illustrate aspects of the book, and Liz
Owen at Garland Science, Taylor & Francis Group for her persistence in ensuring that we
kept working on this volume and finished at least close to one of the deadlines!
An introduction to PCR

1.1 Introduction: PCR, a ‘DNA photocopier’

1
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 everyday molecular biology reagents
to make large numbers of copies of a specific DNA fragment in a test-tube.
PCR has been called a ‘DNA photocopier’. While the concept is simple, PCR
is a complicated process with many reactants. The concentration of
template DNA is initially very low but its concentration increases dramatic-
ally as the reaction proceeds and the product molecules become new
templates. Other reactants, such as dNTPs and primers, are at concentra-
tions that hardly change during the reaction, while some reactants, such
as DNA polymerase, can become limiting. 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 manipu-
lation and analysis.
In the relatively 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. Using PCR we can now isolate essentially any gene from any
organism. It has become a cornerstone of genome sequencing projects, used
both for determining DNA sequence data and for the subsequent study of
putative genes and their products by high throughput screening method-
ologies. Having isolated a target gene we can use PCR to tailor its sequence
to allow cloning or mutagenesis or we can establish diagnostic tests to
detect mutant forms of the gene. PCR has become a routine laboratory tech-
nique whose apparent simplicity and ease of use has allowed nonmolecular
biology labs to access the power of molecular biology. There are many
scientific papers describing new applications or new methods of PCR. Many
commercial products and kits have been launched for PCR applications in
research and for PCR-based diagnostics and some of these will be discussed
in later chapters.

1.2 PCR involves DNA synthesis

PCR copies DNA in the test-tube and uses the basic elements of the natural
DNA synthesis and replication processes. In a living cell a highly complex
system involving many different proteins is necessary to replicate 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 comple-
2 PCR

mentary ‘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 complementary 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 high
fidelity, which means with few mistakes, and in a tightly regulated manner.
DNA synthesis by a DNA polymerase must be ‘primed’, meaning we need
to supply a short DNA sequence called a primer that is complementary to
a template sequence. Primers are synthetically produced DNA sequences
usually around 20 nucleotides long. The DNA polymerase will add
nucleotides to the free 3′-OH of this primer according to the normal base
pairing rules (Figure 1.1).

T G
T G C
dNTPs C
A C G A
A
Primer
DNA T
5' 3' polymerase

3' 5'

Template
T
A C
G
G
5' 3'
A G G T

T C C A C
3' 5'

Synthesis of new DNA strand

5'
A G G T A C T A

T C C A T G A T
3'

Figure 1.1
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) according to base pairing rules.
 An introduction to PCR 3

PCR requires only some of the components of the complex replication

machinery to copy short fragments of DNA in a simple buffer system in a
test tube. Unwinding of the DNA in the cell uses a multi-component
complex involving a variety of enzymes and proteins, but in PCR this is
replaced simply by a heating step to break the hydrogen bonds between
the base pairs of the DNA duplex, a process called denaturation.
Following template denaturation two sequence-specific oligonucleotide
primers bind to their complementary sequences on the template DNA
strands according to normal base pairing rules (Figure 1.2). These primers
define the region of template to be copied. DNA polymerase then begins
to add deoxynucleotides to the 3′-OH group of both primers producing new
duplex DNA molecules (Figure 1.2). This requirement of DNA polymerases
to use primers to initiate DNA synthesis is critical for the PCR process since
it means we can control where the primers bind, and therefore which
region of DNA will be replicated and amplified. If the DNA polymerase was
like an RNA polymerase that does not require a primer then we would have
no way of defining what segment of DNA we wanted to be copied.
At the next heating step the double-stranded molecules, which are
heteroduplexes containing an original template DNA strand and a newly
synthesized DNA strand produced during the first DNA synthesis reaction,
are now denatured. Each DNA single strand can now act as a template for
the next round of DNA synthesis. As discussed in detail in Chapter 2, it is
during this second cycle of PCR that the first DNA single strand of a length
defined by the positions of the primers can be formed. In cycle 3 the first
correct length double-stranded PCR products are formed. In subsequent
cycles there is then an exponential increase in the number of copies of the
‘target’ DNA sequence; theoretically, the number of copies of the target
sequence will be doubled at each PCR cycle. 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. Of course the process is not
100% efficient, and it is usually necessary to carry out more reaction cycles,
often 25 to 40 depending upon the concentration of the initial template
DNA, its purity, the precise conditions and the application for which you
require the product. The specificity and efficiency of PCR, however, means
that very low numbers of template molecules present at the start of the PCR
can be amplified into a large amount of product DNA, often a microgram
or more, which is plenty for a range of detailed analyses. Of course, this
ability to amplify also means that if you happen to contaminate your
reaction with a few molecules of product DNA from a previous reaction, you
may get a false result. This is why performing control reactions is so impor-
tant and we will deal with such contamination problems in Chapter 4.

1.3 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 denature (separate) the strands of the DNA
template. The temperature is then lowered to allow the primers to anneal
by base pairing to their complementary sequences on the template strands;
this temperature varies depending on the primers (see details in Chapter 3).
4 PCR

5' 3'
T G T C G A C T G G A A C A C

A C A G C T G A C C T T G
T G
3' 5'
DNA denatured and
primers annealed

5' 3'
T G T C G A A C A C

3' 5'

Primer 2

T G
T G C
dNTPs C
A C G A
A
Primer 1 T
5' 3'

A C A G C T T G T G
3' 5'

5' 3'
T G T C G A C T G G A A C A C

A C A G C T G A C C T T G
3' 5'
Synthesis of new DNA strands defined by primers
5' 3'
G T C G A C T G G A A C A C

A C A G C T G A C C T T G T G
3' 5'

Figure 1.2
The first cycle of a PCR. A double-stranded template molecule is denatured.
Primers anneal to their complementary sequences on the single-stranded
template. DNA synthesis is catalyzed by a thermostable DNA polymerase. The
result of this PCR cycle is that two copies of the target sequence have been
generated for each original copy.

The annealing temperature is important to ensure high specificity in the

reaction; generally the higher the annealing temperature the more specific
will be the reaction. A temperature of 55°C is commonly used, but in many
cases a higher temperature is better and this can even be as high as 72°C
for some experiments, leading to a two-temperature PCR cycle. Finally, for
 An introduction to PCR 5

efficient DNA synthesis, the temperature is adjusted to be optimal for the

DNA polymerase activity, normally 72°C (see Chapter 3). To amplify the
target DNA it is necessary to cycle through these temperatures several times
(25 to 40 depending on the application). Conveniently, this temperature
cycling is accomplished by using a thermal cycler, a programmable instru-
ment that can rapidly alter temperature and hold samples at the desired
temperature for a set time. This automation is one of the important
advances that led to PCR becoming widely accessible to many scientists and
is covered in more detail in Chapter 3. Before thermal cyclers became avail-
able, PCR was performed by using three water baths set to temperatures of
typically 95°C, 55°C and 72°C, and reaction tubes in racks were moved
manually between the baths.
The other major technological advance that preceded the development
of thermal cyclers was the replacement of DNA polymerase I Klenow
fragment with thermostable DNA polymerases, such as Taq DNA poly-
merase, which are not inactivated at the high denaturation temperatures
used during PCR. The ability to carry out the reaction at high temperatures
enhances the specificity of the reaction (Chapter 4). At 37°C, where
Klenow works best, primers can bind to nontarget sequences with weak
sequence similarity, because mismatches between the two strands can be
tolerated. This leads to poor specificity of primer annealing and the amplifi-
cation of many nontarget products. The introduction of thermostable DNA
polymerases also reduced the cost of a reaction by reducing the amount of
polymerase required. With Klenow, at each denaturing step the enzyme
was also denatured and therefore a fresh aliquot had to be added at each
cycle. Thermostable polymerases retain their activity at the denaturation
temperatures and therefore only need to be added at the start of the
reaction.

1.4 PCR applications and gene cloning

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. It has
become an indispensable tool for routine and repetitive DNA analyses such
as diagnosis of certain genetic diseases within clinical screening laboratories
where speed and accuracy are important factors, and also for sample
identification in forensic and environmental testing. In particular PCR has
become a central tool in the analysis and exploitation of genome sequence
information, for example in gene knockout through RNA interference
where PCR allows the rapid generation of appropriate constructs. It also
facilitates measurement of levels of gene expression by ‘real-time’ PCR that
monitors the level of product amplification at each cycle of the PCR
(Chapter 9), providing information on the relative concentrations of
template cDNA.
In some cases PCR provides an alternative to gene cloning, but in other
cases it provides a complementary tool. In gene cloning a fragment of DNA
is joined by ligation to a cloning vector which is able to replicate within a
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