Series Advisors:

Rob Beynon UMIST, Manchester, UK

Chris Howe Department of Biochemistry, University of Cambridge,
Cambridge, UK
Monoclonal Antibodies
PCR
Forthcoming titles
Separating Cells
Analyzing Chromosomes
Biological Centrifugation
Gene Mapping
Reconstructing Molecular Evolutionary Trees
 PCR

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
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 Contents

Abbreviations x
Preface xii

Chapter 1 An introduction to PCR 1

1. Introduction: PCR, a ‘DNA photocopier’ 1
2. History of PCR 2
3. PCR involves DNA replication 4
4. PCR is controlled by heating and cooling 5
5. Does PCR replace gene cloning? 7
6. PCR applications 7
Chapter 2 Understanding PCR 9
1. How does PCR work? 9
2. PCR: a molecular perspective 11
3. The kinetics of PCR 13
4. Getting started 18
5. Post-PCR analysis 18
Protocol 2.1: Basic PCR 19
Chapter 3 Reagents and instrumentation 22
1. Technical advances in PCR 22
2. Reagents 22
3. PCR buffers 23
4. Nucleotides 24
5. PCR premixes 26
6. Oligonucleotide primers 26
7. DNA polymerases for PCR 35
8. Early PCR experiments 36
9. Thermostable DNA polymerases 37
10. Properties of Taq DNA polymerase 39
vi

11. Thermostable proofreading DNA polymerases 45

12. Tth DNA polymerase has reverse transcriptase activity 48
13. Red polymerases and reagents 49
14. Polymerase mixtures: high-fidelity, long-range and RT PCRs 50
15. Nucleic acid templates 53
16. Mineral oil 56
17. Plasticware and disposables 57
18. Automation of PCR and thermal cyclers 57
Protocol 3.1: Phosphorylation of the 5′-end of an oligonucleotide 66
Protocol 3.2: Extraction of genomic DNA from plant tissue 67
Protocol 3.3: DNA template preparation 68
Protocol 3.4: DNA extraction by proteinase K digestion 69
Chapter 4 Optimization of PCR 71
1. Introduction 71
2. Improving specificity of PCR 72
3. Template DNA preparation and inhibitors of PCR 81
4. Nested PCR improves PCR sensitivity 83
5. Contamination problems 83
6. Preventing contamination 88
7. Trouble-shooting guide 90
Chapter 5 Analysis, sequencing and in vitro expression of PCR 96
products
1. Introduction 96
2. Analysis of PCR products 96
3. Verification of initial amplification product 98
4. Direct DNA sequencing of PCR products 102
5. Direct labeling of PCR products and homogenous assays 115
6. Real time approaches for PCR product detection 118
7. In vitro expression of PCR products 122
Protocol 5.1: Southern blotting 125
 vii

Protocol 5.2: Direct sequencing of a PCR product 126

Protocol 5.3: Cycle sequencing—PE Biosystems ABI Dye terminators 127
Chapter 6 Purification and cloning of PCR products 130
1. Introduction 71
2. Purification of PCR products 131
3. Cloning of PCR products 135
4. PCR cloning vectors 136
5. PCR cloning vector systems 139
6. Analysis of PCR-cloned DNA fragments 146
Protocol 6.1: Blunt-end polishing of PCR fragments 148
Protocol 6.2: Ethanol precipitation of DNA 149
Protocol 6.3: PCR screening of bacterial colonies or cultures 150
Chapter 7 PCR mutagenesis 152
1. Introduction 71
2. Introducing mutations by splicing by overlap extension 153
(SOEing)
3. Deletion mutagenesis 157
4. Insertion mutagenesis 158
5. Point mutations by SOEing 163
6. Inverse PCR mutagenesis 166
7. Unique sites elimination 171
8. Random mutagenesis 172
9. PCR misincorporation procedures 174
10. Recombination strategies 175
11. Gene synthesis 180
Protocol 7.1: Splicing by overlap extension (SOEing) 182
Protocol 7.2: ‘Sticky-feet’ mutagenesis 183
Protocol 7.3: Inverse PCR mutagenesis of a plasmid 185
Protocol 7.4: DNA shuffling 186
viii

Protocol 7.5: Gene synthesis 189

Chapter 8 Analysis of gene expression 192
1. Introduction 192
2. Reverse transcriptase PCR (RT-PCR) 192
3. Semi-quantitative and quantitative RT-PCR 197
4. One-tube RT-PCR 203
5. Differential display 203
6. PCR in a cell: in situ RT-PCR 207
Protocol 8.1: Reverse transcriptase reaction 216
Protocol 8.2: Preparing a polyacrylamide gel 217
Protocol 8.3: Recovery of DNA from the dried gel 220
Protocol 8.4: Attachment of tissues to slides 221
Chapter 9 Cloning genes by PCR 223
A. Cloning genes of known DNA sequence 223
1. Using PCR to clone expressed genes 223
2. Expressed sequence tags (EST) as cloning tools 227
3. Rapid amplification of cDNA ends (RACE) 228
B. Isolation of unknown DNA sequences 230
4. Inverse polymerase chain reaction (IPCR) 231
5. Multiplex restriction site PCR 234
6. Vectorette PCR 235
7. Thermal asymmetric interlaced PCR (TAIL-PCR) 238
8. Linear PCR and the use of biotin/streptavidin 239
9. Degenerate primers based on peptide sequence 240
10. Long-range PCR 244
Protocol 9.1: 5′-RACE 246
Protocol 9.2: Inverse PCR from plant genomic DNA 248
Protocol 9.3: Linear PCR and biotin/streptavidin to isolate regions adjacent250
to a known DNA sequence
Chapter 10 Genome analysis 252
 ix

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

M-MLV Moloney murine leukemia virus

MPSV mutations, polymorphisms and sequence variants
mrPCR multiplex restriction site PCR
MVR minisatellite variant repeat
NBT nitro blue tetrazolium
NF nonfluorescent
nt nucleotides
ORFs open reading frames
PAGE polyacrylamide gel electrophoresis
PASA PCR amplification of specific alleles
PBS phosphate buffered saline
PCR polymerase chain reaction
RACE rapid amplification of cDNA ends
RAPD random amplified polymorphic DNA
RAWIT RNA amplification with in vitro translation
RAWTS RNA amplification with transcript sequencing
RFLP restriction fragment length polymorphism
RT reverse transcriptase
SDS sodium dodecyl sulfate
SNPs single nucleotide polymorphisms
SOEing splicing by overlap extension
SPA scintillation proximity assay
SSCP single strand conformation polymorphism analysis
STEP staggered extension process
STR short tandem repeats
TAIL-PCR thermal asymmetric interlaced PCR
TBR -bipyridine)
tris (2,2′ruthenium (II) chelate
TCA trichloroacetic acid
TdT terminal deoxynucleotidyltransferase
TEMED ,N′-tetramethylenediamine
N,N,N′
TET ,7-tetrachloro-6-carboxy
4,7,2′ fluorescein
TK thymidine kinase
TNF tumor necrosis factor
UNG uracil N-glycosylase
VNTR variable number tandem repeats
 Preface

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

from Applied Biosystems, a division of PE Biosystems. Taq DNA polymerases

are now available from around 25 licensed manufacturers. This means that the
PCR should not be performed using other sources of Taq polymerase, or other
thermal cycler instruments. Some non-licensed manufacturers sell their own
version of thermostable polymerases; however their uses are restricted to
purposes other than PCR, for example DNA sequencing. If you are engaged in
PCR-based testing either for third parties or internal quality control purposes, it
is necessary to obtain a specific licence. In some cases these can be obtained
through the purchase of specialized field test kits. If no kits exist for your
applications then a specific application license should be sought. Clarificiation of
issues associated with the licensing of PCR reagents and thermal cyclers can be
found at http://www.pebio.com/ab/pcrlicensefaq/. Legal processes take some
time to resolve and at the time of writing Promega Corporation has successfully
challenged one of the original patents that was granted for Taq DNA
polymerase, but Roche has been granted leave to appeal. The final outcome of
appeals may have interesting consequences for the companies and users of this
DNA polymerase.

2.1
Key milestones in the development of PCR

1983 Kary Mullis of Cetus Corp invented the PCR.

1985 First paper describing the PCR using the Klenow fragment of DNA
polymerase I (Saiki et al., 1985).
1986 Cetus Corp and Perkin Elmer Corp establish a joint venture company
(Perkin Elmer Cetus) to develop both instruments and reagents for the
biotechnology research market.
1987 Cetus develop a partnership with Kodak for PCR-based diagnostics,
but Kodak terminate this agreement and Hoffman-La Roche become
the new partner.
1988 First paper describing the use of Taq DNA polymerase in the PCR
(Saiki et al., 1988).
1990 Cetus licence certain reagents companies, including Promega,
Stratagene, USB, Pharmacia, Gibco-BRL and Boehringer, to sell
native Taq DNA polymerase for non-PCR applications.
1991 Cetus wins a court case against DuPont who challenged the Cetus
PCR patents.
1991 Perkin Elmer Cetus joint venture dissolved as the PCR rights are
acquired by Hoffman-LaRoche.
1991 Perkin Elmer form a ‘strategic alliance’ with Roche to sell PCR
reagents in the research market. Roche continue to develop the
diagnostics reagents business. Perkin Elmer assume total
responsibility for the thermal cycler business.
4 PCR

1991 Cetus is acquired by Chiron Corp. for non-PCR business aspects, in

particular interleukin-2-based pharmaceuticals.
1992 Roche file a law suit against Promega for alleged infringement of
their license to sell native Taq polymerase for non-PCR applications.
Action also taken against several smaller companies for selling Taq
DNA polymerase without license agreements.
1993 Perkin Elmer merges with Applied Biosystems. The Applied
Biosystems Division of Perkin Elmer assumes responsibility for all
DNA processes such as DNA synthesis, sequencing and the PCR in
addition to the other products associated with protein sequencing and
analysis.
1993 License granted to Boehringer-Mannheim to supply reagents,
including Taq polymerase for use in the PCR.
1993 Kary Mullis, inventor of PCR, wins a Nobel Prize for Chemistry.
1993+ Widespread licensing of PCR technology and Taq DNA polymerases
to a large number of biological supplies companies.
1998 Promega Corporation challenge the original patents on native Taq
DNA polymerase and court proceedings continue.

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

1 Chapter 1 An introduction to PCR

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.
2 Chapter 2 Understanding PCR

Chien, A., Edgar, D.B. and Trela, J.M. (1976)

Deoxyribonucleic acid polymerase from the extreme
thermophile Thermus aquaticus. J. Bacteriol. 127,
1550–1557.

Huang, M.M., Arnheim, N. and Goodman, M.F. (1992) Extension

of base mispairs by Taq DNA polymerase: implications for
single nucleotide discrimination in PCR. Nucleic Acids Res.
20, 4567–4573.

Mullis, K. and Falcona, F. (1987) Specific synthesis of DNA

in vitro via a polymerasecatalyzed chain reaction. Methods
Enzymol. 155 335–350. Protocol 2.1 Basic PCR Equipment Ice
bucket Microcentrifuge Thermal cycler Gel electrophoresis
tank

Materials and reagents Thermostable DNA polymerase and

accompanying buffer (e.g. Taq DNA polymerase) 2 mM dNTP
solution Oligonucleotide primers Template DNA Mineral oil
0.8% agarose (100 ml; 0.8 g agarose in 100 ml 1×TAE) 50×TAE
(242 g Tris base and 57.1 ml glacial acetic acid dissolved
in a final volume of water to give 900 ml, then add 100 ml
0.5 M EDTA, pH 8.0)

Protocol

1. Add the following components to a 0.5 ml microcentrifuge

tube: (a) 5 µl 10×PCR buffer (supplied with enzyme); (b) 5
µl 2mM dNTPs; (c) 1 μ l primer 1 (25 pmol μ l −1 ) (d) 1 μ
l primer 2 (25 pmol μ l −1 ); (e) template DNA (~0.1 pmol
of plasmid to 1 μ g genomic DNA); (f) thermostable
polymerase (1 unit); (g) water to 50 μ l. Ensure that fresh
pipette tips are used for each component and make additions
to fresh sections of the sides of the tube to prevent
mixing of components until all reagents are added. If you
are setting up several reactions then prepare a premix of
any common components to reduce pipetting steps and
potential contamination. Set up control tubes in the same
way but leaving out either DNA or primers.

2. Mix the reagents by centrifuging in a microcentrifuge

for 1 s. Place on ice.

3. If the thermal cycler does not have a heated lid, add 50

μ l of light mineral oil to prevent evaporation during
thermal cycling.

4. Place the tube in a thermal cycler and program for the

following temperature regime: (a) 94°C for 5 min (to
denature the template); (b) 94°C for 1 min; (c) 55°C for 1
min; (d) 72°C for 1 min; (repeat (b)–(d) 25–40 times; (e)
72°C for 2 min (to ensure all molecules are completely
synthesized). The number of cycles depends upon the
complexity and amount of template added. Generally for
plasmid templates 25 cycles is sufficient, whereas for
genomic DNA between 30 and 40 cycles are usually necessary.
It is sometimes helpful during a genomic amplification to
remove 5 μ l aliquots at 30 and 35 cycles to compare with
the 40 cycle sample to follow the accumulation of the
specific band.

5. Samples can be left in the thermal cycler and held at

room temperature or 4° C until you are able to remove them
for further processing. Generally room temperature is
sufficient, although some protocols may require a lower
temperature. It is not a good idea to routinely cool the
samples at 4°C for extended periods if this is not
necessary, as this will reduce the lifetime of the thermal
block in the thermal cycler.

6. Remove the tube from the thermal cycler. If the samples

are overlaid with mineral oil, carefully insert a pipette
tip under the layer of mineral oil and remove about 45 µl
of the reaction, taking care not to remove any mineral oil.
PCR

7. Wipe the outside of the pipette tip with tissue to

remove mineral oil sticking to the tip, then transfer into
a fresh tube.

8. Analyze between 5 and 15 μ l of the sample on an agarose

gel using suitable DNA molecular size markers, as described
in Chapter 5.

Notes

1. The denaturation temperature in step 4(b) should be as

low as reasonably possible to denature the template DNA and
often 92°C will be efficient, although most protocols will
recommend 94°C and most people use this temperature. For
difficult templates, such as GC-rich sequences, a higher
temperature may be necessary, perhaps 96°C. Also this
extended initial denaturation phase may not be necessary or
could be significantly reduced to 1 or 2 min in many
applications. These measures will extend the functional
life of the DNA polymerase molecules.

2. The length of incubation time at each of steps 4(b)–(d)

will depend critically on the thermal cycler
characteristics. Short times of 30 s are often sufficient
and, in robust PCR screening with thermal cyclers that
monitor tube temperature (Chapter 3), the incubations can
be as short as 1 s.

3. The temperature in step 4(c) is a useful starting point

for many PCRs, but can optimally be between 40 and 72°C,
depending upon the primer/template combination.
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5′-end of an oligonucleotide Equipment 1.5 ml
microcentrifuge tubes Adjustable heating block or water
bath Ice bucket

Materials and reagents PCR Polynucleotide kinase and

accompanying buffer (e.g. New England BioLabs) 10 mM ATP
solution Oligonucleotide primer

Protocol

1. Mix the following reagents in a microcentrifuge tube: ′

2 μ l 10×kinase buffer (supplied with enzyme); ′ 5 μ l
primer (20 pmol μ l −1 ); ′ 2 µl 10 mM ATP; ′ 1 μ l
polynucleotide kinase (5–10 units); ′ 10 μ l water.

2. Incubate at 37°C for 30 min.

3. Inactivate the kinase by heating to 90°C for 5 min.

4. Store on ice if to be used immediately, or store at

−20°C for later use. Protocol 3.2 Extraction of genomic DNA
from plant tissue Equipment Pestle suitable for grinding
tissue in a microcentrifuge tube 1.5 ml microcentrifuge
tubes Ice bucket Microcentrifuge

Materials and reagents Sample of plant tissue in a

microcentrifuge tube Liquid nitrogen Extraction buffer (100
mM Tris-HCl, pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl and 10
mM ′ -mercaptoethanol added immediately before use) 20%
sodium dodecyl sulfate (SDS) 5 M potassium chloride (KCl)
Isopropyl alcohol 3 M sodium acetate (NaOAc; pH 5.2) 70%
ethanol sterile ultrapure water

Protocol

1. Grind plant tissue in liquid nitrogen in a 1.5 ml

microcentrifuge tube.

2. Add 500 µl of extraction buffer.

3. Add 35 µl of 20% SDS and incubate tube at 65°C for 5 min.

4. Add 130 μ l 5 M KCl and place tube on ice.

5. Centrifuge at 13000 g for 10 min.

6. Precipitate supernatant at −20°C for 10 min after the

addition of 650 µl isopropyl alcohol and 60 µl 3 M NaOAc.

7. Spin 13000 g for 10 min.

8. Wash pellet with 70% ethanol.

9. Resuspend in 20 μ l sterile water. Protocol 3.3 DNA

template preparation A. Soft tissue or blood rapid
extraction Equipment 1.5 ml microcentrifuge tubes
Microcentrifuge

Materials and reagents Sterile phosphate buffered saline

(1×PBS: 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH
2 PO 4 per liter with pH adjusted to 7.4 with HCl and
autoclaved 15 lb in 2 , 20 min) 0.1 M sodium hydroxide
(NaOH)

Protocol

1. Transfer approximately 10 mg tissue, 30 µl whole blood,

a 2.5 mm blood spot into a 1.5 ml microcentrifuge tube and
adjust volume to 100 μ l with PBS.

2. Add 100 μ l 0.1 M NaOH, close lid, puncture with a

needle and boil for 5 min.

3. Microcentrifuge (13000 g) for 5 min.

4. Transfer supernatant to a fresh tube and use 1–10 μ l

per PCR. PCR

B. Buccal cells Equipment 1.5 ml microcentrifuge tubes 15

ml centrifuge tube Microcentrifuge Bench top centrifuge

Materials and reagents Sterile phosphate buffered saline

(1×PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH
2 PO 4 per liter with pH adjusted to 7.4 with HCl) 0.1 M
sodium hydroxide (NaOH)

Protocol

1. Rinse mouth with water and discard.

2. Rinse mouth vigorously with 10 ml sterile PBS for 10 sec

then collect in 15 ml centrifuge tube

3. Collect cells at 3000g for 10 min in a bench-top

centrifuge.

4. Wash pellet in fresh PBS and repeat centrifugation step.

5. Resuspend cells in 100 μ l PBS and transfer to a

microcentrifuge tube.

6. Add 100 μ l 0.1 M NaOH, close lid, puncture with a

needle and boil for 5 min.

7. Microcentrifuge (13000g) for 5 min.

8. Transfer supernatant to a fresh tube and use 1–10 μ l

per PCR.

Note It is also possible to prepare DNA by boiling samples

for 5–10 minutes without addition of 0.1 M NaOH. Protocol
3.4 DNA extraction by proteinase K digestion Equipment 1.5
ml microcentrifuge tubes Tube tumbler or rotator for gentle
mixing Microcentrifuge

Materials and reagents Liquid nitrogen Sterile phosphate

buffered saline (1×PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na 2
HPO 4 , 0.24 g KH 2 PO 4 per liter with pH adjusted to 7.4
with HCl) Digestion buffer (10 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 25 mM EDTA, 0.5% SDS and 1 mg ml −1 proteinase K
added immediately before use) Phenol Chloroform/isoamyl
alcohol (24:1) 3 M sodium acetate (pH 5.2) Absolute
ethanol, ice cold 70% ethanol, ice cold Tissue paper
Ultrapure water

Protocol Part A Grind fresh tissue to a fine powder using a

mortar, pestle and liquid nitrogen before transferring to a
microcentrifuge tube. For fresh tissue sections, scrape
from a glass slide into a microcentrifuge tube.
Protocol Part B Buccal cells should be prepared as
described in Protocol 3.3B steps 1–4.

1. Add digestion buffer at 0.5 ml per 50 mg tissue and

incubate with rotation at 37°C for 1 h.

2. Add an equal volume of phenol, vortex thoroughly and

separate the phases by centrifugation 13000g for 5 min.

3. Remove the upper aqueous phase ensuring that no

interface material is removed.

4. Re-extract with chloroform/isoamyl alcohol (24:1) and

again remove the upper aqueous phase to a fresh tube.

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.

6. Collect the DNA in a microcentrifuge (13000g, 10 min)

and carefully decant the solution.

7. Wash the pellet in 70% ethanol and centrifuge as in step

6.

8. Decant the ethanol and remove excess by inverting on

tissue paper. Allow to air dry for about 30 min or in a
vacuum desiccator for about 3 min.

9. Resuspend DNA in 100–200 µl water at room temperature

for at least 16 h. Use 1 μ l per PCR. PCR
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Protocol 5.1 Southern blotting Equipment Sandwich box or a
tray Saran wrap or parafilm 3MM paper Blotting paper (paper
towels or special blotting pads) Nitrocellulose membrane
(e.g. Stratagene Duralon-UV™ membrane) UV crosslinker
Material and reagents 20×SSC (3 M NaCl, 0.3 M Na 3
citrate) Depurination solution (0.25 M HCl) Denaturation
solution (1.5 M NaCl, 0.5 M NaOH) Neutralization solution
(1.5 M NaCl, 1 M Tris pH 8) 0.4 M NaOH (optional if no
crosslinker is available)

Protocol

1. If the sizes of the amplified fragments are above 5000

bp then wash the agarose gel twice in depurination solution
for 15 min to break up the fragments into smaller sizes. If
the sizes of the amplified fragments are below 5000 bp then
proceed directly to step 2.

2. Rinse the agarose gel briefly in distilled water.

3. Wash agarose gel in denaturation solution for 30 min

with shaking.

4. Rinse the agarose gel briefly in distilled water.

5. Wash agarose gel in neutralization solution twice for 15

min.

6. Fill a sandwich box or a tray with 20×SSC and make a

platform using a glass plate covered with a wick of three
sheets of 3MM paper soaked in 20×SSC solution.

7. Place the agarose gel on the wick, removing any air

bubbles and surround the gel with Saran wrap or parafilm.

8. Cut a sheet of nitrocellulose membrane to the exact size

of the gel and place on top of the gel, avoiding air
bubbles.

9. Place three wetted sheets of 3MM paper on top of the

membrane.

10. Place absorbent paper towels or blotting pads on top of

the stack, cover with a glass plate and place a weight on
top (250–500 ml of water in a bottle).

11. Transfer overnight.

12. Dismantle the apparatus and crosslink the membrane in a

crosslinker or place on 3MM paper soaked in 0.4 M NaOH for
20 min followed by brief rinsing in 3×SSC

13. Proceed to prehybridization and hybridization steps.

Protocol 5.2 Direct sequencing of a PCR product Equipment
1.5 ml microcentrifuge tubes Adjustable heating block or
water bath

Material and reagents Liquid nitrogen or dry ice Purified

DNA template Oligonucleotide primer 10×sequencing buffer
(depending on the sequencing chemistry of choice)

Protocol

1. Purify PCR product using an appropriate procedure (see

Chapter 6).

2. Mix 0.1–0.5 μ g of purified DNA with 2 pmol primer in 8

µl of water.

3. Heat to 100°C for 5 min. Transfer to liquid nitrogen or

dry-ice for about 1 min until frozen.

4. Thaw at room temperature, add 2 μ l of 10× sequencing

buffer and allow to stand at room temperature for 20 min to
allow annealing of the primer to the template.

5. Perform the sequencing reaction according to the

manufacturer’s instructions for your chosen sequencing
reagent system.

6. Analyze products. Protocol 5.3 Cycle sequencing—PE

Biosystems ABI Dye terminators Equipment Agarose gel
apparatus Microcentrifuge Thermal cycler 0.5 ml
microcentrifuge tubes

Materials Agarose gel Gel loading buffer PCR purification

or gel purification kit Molecular size markers Sequencing
premix (containing buffer, dNTPs, dye terminators,
AmpliTaqFS) Sequencing primer (2 pmol μ l −1 ) Mineral oil
(if no heated lid) Phenol/chloroform from PE Biosystems at
room temperature (it is critical that this is of the
highest quality to avoid fluorescent contaminants that
otherwise affect the sequencing gel) 3 M sodium acetate, pH
5.2 Ethanol, ice-cold 95% and 70%

Protocol

1. Purify the PCR product either by the use of a PCR

clean-up kit or by isolation from an agarose gel (Chapter
6). Resuspend the DNA in a small volume, <30 µl, of elution
buffer.

2. Analyze 0.1 vol of the DNA on an agarose gel containing

1 μ g ml −1 ethidium bromide, together with a known
quantity of molecular size markers. Estimate the quantity
of sample DNA by comparing the relative intensity of the
ethidium bromide stained band with the marker bands. A
total of 30–90 ng of DNA is recommended for direct
sequencing of PCR products.

3. Combine in a 0.5 ml microcentrifuge tube: ′ 30–90 ng

template DNA; ′ 1.6 µl (3.2 pmol) primer; ′ 8 μ l
sequencing premix; ′ xμ l distilled water to give a total
volume of 20 µl. If necessary overlay the tubes with a drop
of mineral oil.

4. Place in a thermal cycler and subject to 25 cycles of

the following: ′ 96°C, 30 s; ′ 50°C, 15 s; ′ 60°C, 4 min.

This takes approximately 3 h to complete.

5. Add 80 µl water to the 20 μ l sequencing reaction. If

mineral oil was used, pierce the oil layer with a pipette
tip and remove 95 µl of sequencing reaction to a fresh
tube.

6. Extract with ABI phenol/chloroform by adding an equal

volume and vortexing for 30 s. Immediately centrifuge the
tubes in a microcentrifuge at 13000 g for 2 min.

7. Carefully transfer 90 µl of the upper aqueous phase to a

fresh microcentrifuge tube. It is critical that no
interface material is carried over as this will interfere
with the fluorescence detection not only of your samples
but of other users whose samples are analyzed on the same
gel. If you are in any doubt about the purity of your
sample re-extract with phenol/chloroform.

8. Add 0.1 vol sterile 3 M sodium acetate, vortex for 1 s,

then add 2.5 vol icecold 95% ethanol, vortex for 1s and
leave on ice for up to 15 min.

9. Pellet the DNA in a microcentrifuge (13000 g),

preferably at 4°C for 15 min. As the pellet may not be
visible (unless you use a pellet paint, Chapter 3), orient
the tubes conveniently with the hinges to the outside to
indicate the side of the tube on which the DNA will be
pelleted.

10. Remove the ethanol by aspirating off with a pipette,

taking care not to touch the side of the tube with the DNA.

11. Wash the pellet by adding 150 μ l ice-cold 70% ethanol,

vortex briefly and centrifuge as before with the tube in
the same orientation.

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.

13. The samples are now ready to be resuspended and

fractionated on an ABI sequencing instrument.
6 Chapter 6 Purification and cloning of
PCR products

Aslanidis, C. and de Jong, P.J. (1990) Ligation-independent

cloning of PCR products (LIC-PCR). Nudeic Acids Res. 18,
6069–6074.

Figure 6.7

PCR screening of bacterial cultures to identify clones

carrying the desired insert. Gel a:

screening for the presence of three different inserts in

lanes 1–3, 4–6 and 7–9. With the

exception of the clone in lane 9 the other clones have

inserts. Gel b: Screening of nine

individual bacterial colonies. Only clones 1, 3, 7 and 8

carry an insert. PCR was

performed according to Protocol 6.3B. Photographs were

kindly provided by A.Neelam.

Clark, J.M. (1988) Novel non-templated nucleotide addition

reactions catalyzed by prokaryotic and eukaryotic DNA
polymerases. Nucleic Acids Res. 16, 9677–9686.

Haun, R.S. and Moss, J. (1992) Ligation-independent cloning

of glutathione Stransferase fusion genes for expression in
Escherichia coli. Gene 112, 37–43.

Haun, R.S., Serventi, I.M. and Moss, J. (1992) Rapid,

reliable ligation-independent cloning of PCR products using
modified plasmid vectors. BioTechniques 13, 515–518.

Kaufman, D.L. and Evans, G.A. (1990) Restriction

endonuclease cleavage at the termini of PCR products.
BioTechniques 9, 304.

Marchuk, D., Drumm, M., Saulino, A. and Collins, F.S.

(1991) Construction of Tvectors, a rapid and general system
for direct cloning of unmodified PCR products. Nucleic
Acids Res. 19, 1154.

Moreira, R.F. and Noren, C.J. (1995) Minimum duplex

requirements for restriction enzyme cleavage near the
termini of linear DNA fragments. BioTechniques 19, 56–59.

Pan, G., Luetke, K., Juby, C.D., Brousseau, R. and

Sadowski, P. (1993) Ligation of synthetic activated DNA
substrates by site-specific recombinases and topoisomerase
I. J. Biol. Chem. 268, 3683–3689.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)

Molecular Cloning: a Laboratory Manual, 2nd edn. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Direct

cloning and sequence analysis of enzymatically amplified
genomic sequences. Science 233, 1076–1078.

Vogelstein, B. and Gillespie, D. (1979) Preparative and

analytical purification of DNA from agarose. Proc. Natl
Acad. Sci. USA 76, 615–619.

Yang, R.C.A., Lis, J. and Wu, B. (1979) Elution of DNA from

agarose gels after electrophoresis. Methods Enzymol. 68,
176–182. Protocol 6.1 Blunt-end polishing of PCR fragments
Materials T7 DNA polymerase 10×T7 DNA polymerase buffer 100
mM MgCl 2 2 mM each dNTP mix Distilled water

Equipment Microcentrifuge tube Microcentrifuge Water bath

or heat block set at 70°C

Protocol

1. Combine in a microcentrifuge tube: ′ purified PCR

product, up to 40 µl; ′ 10×T7 polymerase buffer, 5 µl; ′
100 mM MgCl 2 , 2 μ l; ′ 2 mM dNTP mix, 2.5 µl; ′ T7 DNA
polymerase, 1 unit; ′ Water, up to 50 μ l.

2. Collect reagents and mix by a brief (1 s) centrifugation

step.

3. Incubate at room temperature for 30 min.

4. Heat to 70°C for 10 min to inactivate the polymerase.

Protocol 6.2 Ethanol precipitation of DNA Materials 3 M
sodium acetate (pH 5.2) 95% ethanol 70% ethanol 10 mM
Tris-HCI (pH 8.0)

Equipment Microcentrifuge tube Microcentrifuge Vortex mixer

(optional) Ice bucket (optional) Tissue paper Vacuum
dessicator (optional)

Protocol

1. Measure the volume of DNA solution.

2. Add 0.1 vol of 3 M sodium acetate (pH 5.2).

3. Mix the sample by inversion of using a vortex mixer for

2 s.

4. Add 2.5 vol of 95% ethanol at room or ice temperature

and mix by inversion or on a vortex mixer.

5. Incubate at room temperature or in an ice bath for 15–60

min.

6. Centrifuge for 15 min at 13000 g in a microcentrifuge at

4°C. Orient the tubes to allow identification of the pellet
position after centrifugation.

7. Carefully discard the liquid either by decanting or

using a Pipetman (Gilson). Touch the tube onto tissue paper
to remove excess liquid.

8. Add 0.5–1.5 ml ice-cold 70% ethanol and repeat steps 6

and 7.

9. Briefly allow the pellet to dry either by allowing the

tube to stand inverted on tissue paper, or by placing in a
vacuum desiccator for 2–3 min. The intention is to remove
traces of ethanol, but overdrying should be avoided
otherwise dissolving the pellet becomes difficult.

10. Dissolve the DNA precipitate in an appropriate volume

of sterile water or 10 mM Tris-HCl (pH 7.5–8.5). Protocol
6.3 PCR screening of bacterial colonies or cultures
Materials PCR reactant mix containing 25 pmol each primer,
0.2 mM each dNTP, 1×PCR buffer, 0.5 unit Taq DNA polymerase
and water. LB or 2×TY culture medium containing the
appropriate antibiotic for plasmid growth Agarose gel

Equipment 0.5 or 0.2 ml tubes or microtiter plates for PCR

Microcentrifuge Orbital incubator for growth of bacterial
cultures

Protocol Part A—from colonies It is possible to directly

analyze whether colonies contain a desired

recombinant plasmid and at the same time to set up cultures

from the colonies

being tested to allow subsequent isolation of recombinant

plasmids from

appropriate cultures.
1. Pipette a 20 μ l aliquot of a PCR reactants premix into
a PCR tube.

2. Touch a toothpick to the edge of the colony, taking care

not to transfer a visible amount of the colony. Swirl the
toothpick briefly in the PCR mix and then if required
inoculate a tube containing growth medium such as 2×TY or
LB, to allow growth of a culture, or streak onto an
appropriate agar plate.

3. Collect and mix reagents by a brief (1 s) centrifugation

step.

4. Perform the PCR using the following conditions: ′ 94°C,

5 min to bring about cell lysis; ′ 94°C, 30 s; 55°C, 30 s;
72°C, 30 s for 25–30 cycles; ′ 72°C, 1 min.

5. Analyze a 5 µl aliquot on an agarose gel.

6. Meanwhile place the inoculated cultures in an orbital

incubator at 37°C to allow growth of the culture for
subsequent plasmid isolation from PCR positive colonies, or
incubate the agar plate to allow subsequent culture
inoculation once positives have been identified.

Protocol Part B—from cultures An alternative approach is to

grow overnight cultures from colonies and then

test these by PCR to determine which to use for plasmid

isolation.

1. Transfer a 5 μ l aliquot of culture to a 0.5 ml PCR tube

and incubate at 99°C for 5 min.

2. Centrifuge at 13000 g in a microcentrifuge for 5 min.

3. Transfer a 1 μ l aliquot of the supernatant to a PCR

tube containing 19 μ l of premix.

4. Collect and mix reagents by a brief (1 s) centrifugation

step.

5. Perform the PCR using the following conditions: ′ 94°C,

3 min; ′ 94°C, 30s; 55°C, 30s; 72°C, 30 s for 25–30 cycles;
′ 72°C, 1min.

6. Analyze a 5 µl aliquot on an agarose gel.

7. Prepare plasmid DNA from cultures showing positive PCR

products.

Notes A hot start procedure (Chapter 4) will enhance

specificity, but the short cycle

times often used make this approach unsuitable for TaqStart

antibody

approaches. The length of each incubation step in steps 4

(Protocol A) and 5 (Protocol B)

depends upon the type of thermal cycler. In some cases for

reaction temperature

monitoring instruments the time may be as short as 1–10 s.

The annealing temperature depends upon the length of the
primers and should

be as high as possible to ensure specificity of priming. It

is worth testing a

positive culture at different annealing temperatures.This

can conveniently be

undertaken with a gradient thermal cycler.

7 Chapter 7 PCR mutagenesis

Cadwell, R.C. and Joyce, G.F. (1992) Randomization of genes

by PCR mutagenesis. PCR Methods Applic. 2, 28–33.

Clackson, T. and Winter, G. (1989) ‘Sticky feet’-directed

mutagenesis and its application to swapping antibody
domains. Nucleic Acids Res. 17, 10163–10170.

Clackson, T., Güssow, D. and Jones, P.T. (1991) General

applications of PCR to gene cloning and manipulation. In:
PCR1: a Practical Approach, pp. 187–214 (McPherson, M.J.,
Taylor, G.R. and Quirke, P, eds.). Oxford University Press,
Oxford.

Crameri, A., Railland, S.-R., Bermudez, E. and Stemmer,

W.P.C. (1998) DNA shuffling of a family of genes from
diverse species accelerates directed evolution. Nature 391,
288–291.

Hemsley, A., Arnheim, N., Toney, M.D., Cortopassi, G. and

Galas, D.J. (1989) A simple method for site-directed
mutagenesis using the polymerase chain reaction. Nucleic
Acids Res. 17, 6545–6551.

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.

Hubner, P., lida, S. and Arber, W. (1988) Random

mutagenesis using degenerate oligodeoxyribonucleotides.
Gene 73, 319–325.

Moore, J.C., Jin, H.-M., Kuchner, O. and Arnold, F.H.

(1997) Strategies for the in vitro evolution of protein
function: enzyme evolution by randon recombination of
improved sequences. J. Mol. Biol 272, 336–347.

Stemmer, W.P. (1994) Rapid evolution of a protein in vitro

by DNA shuffling. Nature 370, 389–391.

Zaccolo, M., Williams, D.M., Brown, D.M. and Gherardi, E.

(1996) An approach to random mutagenesis of DNA using
mixtures of triphosphate derivatives of nucleoside
analogues. J. Mol. Biol. 255, 589–603.

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

Materials and reagents Plasmid template PCR reagents

Oligonucleotide primers Ethanol 1 M Tris-HCl pH8.0 0.8%
agarose (100 ml: 0.8 g agarose in 100 ml of 1×TAE)

Protocol Part A—primary PCRs

1. Set up two PCRs to perform primary PCR amplifications of

the two DNA fragments to be spliced. One reaction should
contain primers 1 and 2 and the second primers 3 and 4. PCR
conditions and the reaction mixes should be as described in
Protocol 2.1, except use:

′ 100–500 ng of plasmid template and only 10–15 cycles;

′ an annealing temperature as close to 72°C as possible.

2. Analyze the two amplified products on an agarose gel.

Depending on the purity of the products either:

′ purify using a PCR clean-up kit (Chapter 6) to remove

buffer and primers; or

′ concentrate the samples by ethanol precipitation,

separate through a 0.8% agarose gel, excise the two
fragments and recover from the agarose (Chapter 6).

3. Elute each fragment in a low volume (20–30 μ l) of 10 mM

Tris-HCl (pH 8.0) and estimate the concentration on an
agarose gel using known quantities of appropriate DNA
molecular size markers.

Protocol Part B—secondary PCR

1. Mix the fragments in a 1:1 molar ratio so that the

amount of DNA added to PCR 2 is around 100 ng. Add 25 pmol
each of primers 1 and 4.

2. Perform PCR 2 under the same temperature regime as for

PCR 1 for 10–15 cycles.

3. Size fractionate the final product through an agarose

gel, gel-purify the fragment and clone into a suitable
vector (Chapter 6). Protocol 7.2 ‘Sticky-feet’ mutagenesis
Equipment 0.5 or 0.2 ml PCR tubes Thermal cycler Gel
electrophoresis tank
Materials and reagents Phosphorylated oligonucleotide
primers PCR reagents Phagemid single-stranded template DNA
5 mM dNTP solution Thermostable DNA polymerase and
accompanying buffer (e.g. Perkin Elmer or Boehringer
Mannheim) T7 DNA polymerase and accompanying buffer (e.g.
New England BioLabs.) 100 mM DTT T4 DNA ligase and
accompanying buffer (eg. GiBCO-BRL) 500 mM EDTA pH 8.0 0.8%
agarose (100 ml; 0.8 g agarose in 100 ml of 1×TAE)

Protocol

1. Make sure that the appropriate primer for the primary

PCR amplification contains a 5′-phosphate prior to use.
This can be requested when the primers are synthesized or
you can perform a T4 DNA kinase reaction (Protocol 3.1).

2. Perform a PCR amplification with the two primers, as

described in Protocol 2.1. Use an annealing temperature as
close to the polymerization temperature as possible.

3. Size-fractionate the reaction products through an

agarose gel and purify the ‘sticky-feet’ fragment (Chapter
6).

4. Set up a DNA extension reaction containing:

′ ~100–200 pmol ‘sticky-feet primer’;

′ 200 ng of M13 or phagemid single-stranded template DNA

propagated in a dut − , ung − strain,or alkali-denatured
and neutralized supercoiled plasmid;

′ 5 mM each dNTP;

′ 10×DNA polymerase buffer;

′ 2–5 units of a thermostable proofreading DNA polymerase

(Chapter 3).

5. Incubate the reaction at:

′ 94°C for 2 min;

′ 65°C for 1 min;

′ 37°C for 1 min; with a ramping rate that allows about 1

min between the temperatures, to allow template
denaturation and annealing of the sticky feet fragment.

6. To the reaction add the following components in order:

′ T7 DNA polymerase buffer;

′ 5 mM ATP;

′ 100 mM DTT;

′ 400 units T4 DNA ligase; and

′ 1 unit T7 DNA polymerase.

7. Incubate the reaction at 37°C for 30 min, inactivate

with 1 µl of 500 mM EDTA and transform a dut + , ung + E.
coli strain with one tenth of the final reaction.

8. Screen for mutant plaques (M13) or colonies

(phagemid/plasmid) using PCR amplification and primers
flanking the inserted sequence.While M13 phage are plated
for the production of plaques representing the slower
growth of cells infected by the phage, phagemids can infect
a cell but then replicate as plasmids and carry none of the
phage infection functions. Phagemid transformants are
therefore selected for as colonies capable of growth on
plates containing an antibiotic for which the phagemid
carries a resistance gene. Protocol 7.3 Inverse PCR
mutagenesis of a plasmid Equipment 0.5 or 0.2 ml PCR tubes
Thermal cycler Gel electrophoresis tank

Material and reagents Template plasmid DNA Thermostable DNA

polymerase and accompanying buffer (e.g. Perkin Elmer,
Boehringer Mannheim) Kinased oligonucleotide primers 2 mM
dNTP solution T4 DNA ligase and accompanying buffer (e.g.
GiBCO-BRL) 1 M Tris-HCl pH 8.5 Competent E. coli cells

Protocol

1. Mix in a 0.5 µl microcentrifuge tube:

′ 2 µl 10×polymerase buffer;

′ 2 µl 2 mM dNTPs;

′ 2 µl primer A (kinased; 5 pmol µl −1 );

′ 2 μ l primer B (kinased; 5 pmol ml −1 );

′ 1–10 µl plasmid DNA (1–100 fmol);

′ 1 µl proofreading polymerase (2–3 units);

′ Water to 20 μ l.

2. Thermal cycle through:

′ 95°C for 2 min to denature the template, then 25 cycles of

′ 95°C for 1 min;

′ 55°C for 1 min;

′ 72°C for 5–6 min;

′ and a final incubation at 72°C step for 10 min.

3. Purify the products from an agarose gel using an

appropriate gel purification kit eluting in no more than 30
μ l 10 mM Tris-HCl (pH 8.5) (Chapter 6).

4. Ligate 8–10 µl of DNA in a 12–15 µl reaction containing

T4 DNA ligase buffer including 1 mM ATP and 200 units T4
DNA ligase to catalyze recircularization of the linear
plasmid molecules in a blunt-end ligation reaction.

5. Incubate at room temperature for 4–6 h and transfect

competent cells with 5– 10 μ l of the ligation reaction.

Note

The annealing temperature will depend upon the melting

temperature of the

oligonucleotides and it should ideally be as high as

possible to ensure specificity. Protocol 7.4 DNA shuffling
Equipment 1.5 ml microcentrifuge tubes 0.5 or 0.2 ml PCR
tubes Adjustable heating block or water bath Thermal cycler

Materials and reagents Template DNA 10×DNAse 1 digestion

buffer [400 mM Tris-HCl (pH 7.9), 100 mM NaCl, 60 mM MgCl 2
, 100 mM CaCl 2 ] DNase 1 Ethanol 4% Nusieve GTG agarose
(100 ml; 4 g Nusieve GTG agarose in 100 ml 1×TAE)
Thermostable DNA polymerase and accompanying buffer (e.g.
Perkin Elmer, Boehringer Mannheim) Flanking oligonucleotide
primers 2 mM dNTP solution This procedure describes
shuffling of a single gene or several gene fragments.

Protocol Part A—template DNA

1. Prepare template fragments either by PCR amplification

or by restriction digestion. It is necessary to use
approximately 0.5 μ g of template DNA in a shuffling
reaction. Since only a proportion of the starting DNA will
be fragmented in the correct size range you must ensure you
generate about five times more of each template
(approximately 2.5 μ g per template fragment).

2. Purify the fragments from vector and, if PCR is used, it

is essential to remove the primers. Use an appropriate
purification approach such as a spin column or gel
purification (Chapter 6).

Protocol Part B—DNAse I digestion

3. Mix the following components in a microcentrifuge tube:

′ 5 μ l 10×DNAse 1 digestion buffer;

′ 5 μ g template DNA;

′ 0.5 units DNAse 1;

′ total volume 50 µl.

4. Incubate at room temperature for 15 min.

5. Terminate the reaction by heating to 90°C for 10 min.

6. Ethanol precipitate (Chapter 6).

Protocol Part C—fragment purification

7. Electrophorese the DNA through a 4% NuSeive GTG agarose

gel (BioMolecularWhittaker) usingTAE buffer with EDTA at
0.2 mM.

8. Visualize the ethidium bromide-stained gel under UV

illumination and recover the appropriate size fraction
(normally 50–100 bp, although for small genes recovering a
smaller size range such as 25–50 bp is recommended).

9. Trim the slice to remove excess agarose, but minimize

exposure to UV.

Protocol Part D—DNA shuffling reaction

10. Combine in a 0.5 ml microcentrifuge tube:

′ gel slice maximum 12.5 μ l;

′ 5 μ l 10×Taq DNA polymerase buffer;

′ 5 μ l 2 mM each dNTP;

′ 1–3 units Taq DNA polymerase;

′ total volume 50 µl.

11. Perform thermal cycling (for an instrument monitoring

tube temperature) according to the following scheme:

′ 95°C for 5 mins, then 40–60 cycles of

′ 95°C, 30s;

′ 50°C, 30s;

′ 72°C, 1 min;

′ and a final 72°C, 5 min extension.

12. Purify the final product using an appropriate spin

column method as the reaction contains agarose eluted in
approximately 30 μ l 10 mM Tris-HCl (pH 8.0).

Protocol Part E—final PCR step

13. Combine in a microcentrifuge tube:

′ 1.25 µl shuffled DNA;

′ 5 µl 10×Taq DNA polymerase buffer;

′ 5 µl 0.2 mM each dNTP;

′ 25 pmol each flanking primer;

′ 1 unit Taq DNA polymerase;

′ total volume 50 μ l.

14. Perform thermal cycling under the same conditions as in

step 11 for 20–30 cycles. If insufficient full-length
product is generated then the number of cycles can be
increased.

15. Clone the products into an appropriate expression

vector for subsequent screening or selection. Protocol 7.5
Gene synthesis

This protocol describes the synthesis of a gene of

approximately 300 bp from six
oligonucleotides each 65–70 nt long and with 12–15 nt
terminal overlaps. Equipment Microcentrifuge
Microcentrifuge tubes Thermal cycler Agarose gel system

Materials and reagents Oligonucleotides designed to

generate synthetic gene at 50 pmol μ l −1 Proofreading
thermostable DNA polymerase 10×polymerase buffer 2 mM dNTPs
Water

Protocol Part A—one-tube synthesis

1. Combine the following reagents in a 0.5 ml

microcentrifuge tube:

′ each primer, 5 (µl;

′ 10×buffer, 4 µl;

′ dNTPs, 4 µl;

′ polymerase, 5 units;

′ total volume, 40 μ l.

2. Mix the reactants by a brief (1s) centrifugation step.

3. Place the tube in a thermal cycler preset to 95°C and

incubate for 2 min.

4. Cool the tube to room temperature for 4–5 min, either by

programing the thermal cycler, or by removing the tubes
from the instrument and allowing them to stand at room
temperature.

5. Incubate at 72°C for 5 min.

6. Set up the following reaction:

′ reaction mix from step 5, 5 μ l;

′ 10×buffer, 2.5 µl;

′ dNTPs, 2.5 μ l;

′ Primer 1 (Figure 7.18), 0.5 µl;

′ Primer 2 (Figure 7.18), 0.5 μ l;

′ Polymerase, 5 units;
′ Total volume, 25 μ l.

7. Mix the reactants by a brief (1 s) centrifugation step.

8. Perform the 25–30 cycles of the following PCR conditions:

′ 92°C, 30s;

′ 55°C, 30s;

′ 72°C, 1 min;

′ a final incubation at 72°C for 5 min.

9. Purify the full-length products from an appropriate

concentration agarose gel (Chapter 6).

Protocol Part B—step-wise synthesis

1. Set up three parallel reactions, each containing one

primer pair with reference to Figure 7.18: primer 1+2;
primer 3+4; primer 5+6:

′ primer A (1 or 3 or 5), 5 µl;

′ primer B (2 or 4 or 6), 5 µl;

′ 10×buffer, 1.5 µl;

′ dNTPs, 1.5 µl;

′ polymerase, 5 units;

′ total volume, 15 μ l;

2. Mix the reactions by a brief (1 s) centrifugation step.

3. Incubate according to Procedure A, steps 3–5.

4. Set up the following reaction:

′ primer 1+2 reaction, 5 µl;

′ primer 3+4 reaction, 5 μ l;

′ 10×polymerase buffer, 2 µl;

′ dNTPs, 2 µl;
′ polymerase, 2.5 units;

′ total volume, 20 µl.

5. Incubate according to Procedure A, steps 3–5.

6. Set up the following reaction:

′ primer 1+2+ 3+4 reaction, 5 μ l;

′ primer 5+6 reaction, 5 µl;

′ 10×polymerase buffer, 2 µl;

′ dNTPs, 2 µl;

′ polymerase, 2.5 units;

′ total volume, 20 µl.

7. Incubate according to Procedure A, steps 3–5.

8. Amplify the full-length gene from the reaction mix from

step 7 by following Protocol Part A steps 6–9.
8 Chapter 8 Analysis of gene expression

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Yoshikawa, Y., Mukai, H., Asada, K., Hino, F. and Kato, I.

(1998) Differential display with
carboxy-X-rhodamine-labeled primers and the selection of
differentially amplified cDNA fragments without cloning.
Anal. Biochem. 256, 82–91. Protocol 8.1 Reverse
transcriptase reaction Equipment Adjustable heating block
or water bath Gel electrophoresis tank

Materials and reagents RNA isolation kit (e.g. Qiagen

RNeasy ® minikit) First-strand cDNA synthesis kit (e.g.
ProSTAR™ First-strand RT-PCR Kit, Stratagene) or reverse
transcriptase buffer (AMV 10×RT buffer; 25 mM Tris-HCl pH
8.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl 2 ) 10 mM dNTP mix
Random hexamer, gene-specific or oligo-dT primer RNase
inhibitor Reverse transcriptase (e.g. Qiagen) 0.8% agarose
(100 ml; 0.8 g of agarose in 100 ml of 1×TAE)

Protocol

1. Add to a reaction tube the following in order:

′ 1–5 μ g of RNA (the amount of RNA used for the RT

reaction depends on the abundance of the target
transcript);

′ 1 mM of each dNTP

′ 50–100 pmol of primer (Random hexamers, gene specific

primers, or oligo-dT primers)

′ 1–5 units of an RNase inhibitor

′ Water up to 49 μ l.

2. Heat the reactions to 90°C for 5 min (heating the RNA

eliminates RNA secondary structures and in turn increases
the priming efficiency).

3. Add 50–200 units of reverse transcriptase.

4. Incubate reactions at 42°C for 1h.

5. Heat inactivate the reaction at 90°C for 10 min (heating

the reaction to 90°C inactivates the reverse transcriptase
and denatures remaining RNA-DNA hybrids). Protocol 8.2
Preparing a polyacrylamide gel Equipment Sequencing back
plate, glass plate, spacers and a comb Good-quality tape
(e.g. masking tape) Benchcote and Saran wrap Air-bubble
remover Electrophoresis apparatus and power supply
Adjustable heating block or water bath Gel dryer X-ray film
cassette

Materials and reagents Ethanol Silanizing solution (e.g.

SIGMACOTE ® Sigma catalog no. SL-2 from Sigma Chemicals)
Acrylamide solution and TEMED (e.g. Sigma Chemicals)
Electrophoresis buffer (10×TBE; 0.89 M Tris base, 0.89 M
boric acid, 20 mM EDTA) Loading buffer (6×loading buffer;
0.25% bromophenol blue, 0.25% xylene cyanol FF, 30%
glycerol in water) 3MM paper X-ray film (e.g. Kodak)

Protocol Part A—gel template

1. Clean the glass plates, spacers and comb (spacers and

comb must be of the same thickness) thoroughly using
detergent and rinse extensively with distilled, deionized
water.

2. Dry the plates by wiping with 95% ethanol and allowing

to air dry.

3. Coat the inner surface of the front ‘notched’ plate in a

silanizing solution such as SIGMACOTE ® .

4. Assemble the gel template by placing the spacers onto

the inner surface of the back plate, then place the front
plate with its inner surface down on the spacers. It is a
good idea to leave the spacers overhanging the edges of the
glass plates so that they can be pushed gently into the
correct position between the plates.

5. Seal the sides and bottom of the sandwich using a

good-quality tape. It is critical that the tape does not
leak when the acrylamide solution is added.

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!

1. Cover the bench with an appropriate disposable covering

such as benchcote.

2. Add 40 μ l TEMED to your acrylamide solution and mix by

swirling gently.

3. Pick up the gel sandwich and hold it at about 45° from

the horizontal and balance it on the bottom corner furthest
from your hand. Generally for a righthanded person this
means holding the gel sandwich with your left hand at an
angle of 45° from horizontal and then raising the left side
slightly so that the bottom right corner only is in contact
with the bench.

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.

5. Insert the comb (either a shark-tooth comb or a standard

comb) and lay the gel at an angle of 30° to the horizontal
to allow it to set. If you generate airbubbles whilst
pouring, it is important to keep pouring and then to use an
airbubble remover to eliminate the bubbles. Air-bubble
removers can be obtained from a number of commercial
sources and are basically a piece of long plastic,the same
thickness as the plastic spacers, with a small hook at the
end which ‘traps’ bubbles, which can then be pulled to the
top of the gel and eliminated.

6. Remember if using a glass pipette to empty the pipette

back into the flask, otherwise the acrylamide will set in
the pipette, making it difficult to clean. The acrylamide
in the flask can be examined occasionally to check whether
the acrylamide in the gel has set.

7. Once the gel has polymerized (approximately 60 min at

room temperature), it can either be used directly or stored
at 4°C for 1–2 days sealed in Saran Wrap.

Protocol Part C—running and exposing the gel

1. Remove the tape along the bottom edge of the gel
sandwich and place in the gel in the electrophoresis
apparatus with the notched plate adjacent to the upper
buffer chamber.

2. Fill the upper buffer chamber with 1×electrophoresis

buffer (10×TBE). Remove the comb carefully and rinse the
wells with buffer to wash away trapped acrylamide that may
otherwise polymerize in the wells, leading to distorted DNA
bands.

3. Before loading the samples, prerun the gel at 60 W

(constant) for 30 min so that the polyacrylamide gel
becomes equilibrated and reaches the desired temperature.

4. Mix the DNA samples with loading buffer.

5. Heat the samples to 95°C for 5 min and chill on ice to

denature and prevent re-association of the single-stranded
molecules.

6. Load 3–5 µl of each sample using a gel-loading syringe

or special pipette tips. It is important to keep the sample
volume low to prevent leakage into adjacent wells leading
to ‘ghost bands’.

7. Run the gel at 60 W (constant) until the bromophenol dye

has migrated to the end of the gel.

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.

9. Remove the upper plate by gently forcing the sandwich

apart using a plastic spatula. Try to avoid doing this
around the notch segment of the upper plate as this can
easily break.

10. To transfer the polyacrylamide gel to a sheet of 3MM

paper lay the paper onto the gel and then lift it in a
gentle rolling motion from one end to the other. Do not fix
the gel as for DNA sequencing before transferring because
this will interfere with subsequent re-amplification and
cloning.

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

Materials and reagents Elution buffer [0.5 M ammonium

acetate, 10 mM magnesium acetate, 1 mM EDTA (pH8.0), 0.1%
SDS]

Protocol

1. Locate the selected DNA fragment by aligning the X-ray

film with the dried gel.

2. Cut out the dried gel slice using a sharp razor blade.

3. Crush the gel slice in a microcentrifuge tube, most

conveniently using a disposable pestle, add 2 vol of
elution and incubate for 5–12 h at 37°C.The elution time
depends on the DNA fragment length.The longer the fragment
the longer the incubation time, however we recommend using
a slightly longer time rather than a shorter time.

4. Centrifuge the sample at full speed in a microcentrifuge

for 5 min. Recover the supernatant, add a further aliquot
of elution buffer to the gel and repeat the centrifugation.

5. Combine the supernatants and ethanol precipitate.

Redissolve the DNA in 10–20 µl of TE or sterile distilled
water. Protocol 8.4 Attachment of tissues to slides
Equipment Glass microscope slides Adjustable heating block

Materials and reagents Phosphate-buffered saline (1 liter

of 10×PBS: 80 g NaCl, 2 g KCl, 14.4 g Na 2 HPO 4 , 2.4 g KH
2 PO 4 , pH 7.4 with HCl) Silane solution (e.g. Bind-Silane
solution from Amersham Pharmacia catalog no. A-174)
Paraformaldehyde and xylene (e.g. Sigma Chemicals) 4 M NaOH
solution in a plastic bottle Ethanol Acetic acid Hen
egg/glycerol mixture or poly-lysine (e.g. Sigma Chemicals)

Protocol Part A—single cells and colonies

The attachment of single cells or colonies to glass slides

is very rapid and

simple:

1. For a cell suspension: resuspend cells in 1×PBS and

spray onto silanetreated glass slides.

2. For single cells:

′ resuspend the cells in 1×PBS and transfer a drop to an
untreated glass slide;

′ dry the glass slide for 1–2 minutes at 50°–55°C before

adding the silane solution. This ensures an even
distribution of cells because cells added to silane-treated
glass slides become compressed and concentrated during the
rapid water evaporation;

′ fix the cells in 4% paraformaldehyde and 0.04 M NaOH for

15–30 min at 4°C;

′ wash the glass slide in 1×PBS and incubate in 100%

ethanol for 5 min.

′ permeabilize the cells in xylene for 30 s;

′ rehydrate by transferring the slide through an ethanol

series (100%, 90%, 70%, 50% and 30%) each for 5 min
followed by washing in 1×PBS.

Protocol Part B—attachment of sections

1. Place sections, normally paraffin wax-embedded, in water

drops onto silanecoated glass slides [Bind-Silane solution:
2 μ l Bind-Silane (Pharmacia catalog no. A-174), 2 μ l
acetic acid, 1 ml 100% ethanol), containing a protein (hen
egg/ glycerol) mixture or poly-lysine].

2. Incubate slides at 56°C for 5 min followed by an

incubation at 37°C for 2–3 h.

3. Remove the paraffin-based wax embedding material by

washing for 5 min in xylene, xylene/ethanol (1:1), and 100%
ethanol followed by brief drying.

4. Add 0.5 μ l of Bind-Silane to allow post-xylene

attachment. The use of the protein/glycerol mixture is also
essential to keep the sections attached during the xylene
treatment.

5. Rehydrate the sections through an ethanol series as

described in Part A.

Protocol Part C—attachment of whole organs

1. Place the organs on silane-treated glass slides in 1×PBS

and allow to dry at 37°C.The time will vary depending on
the size and complexity of the organ.
2. Add a fresh aliquot of silane to ensure complete binding
to the glass slide.

3. Fix the organs in 95% ethanol at −70°C for between 2 and

12 h, again depending on the organ size.

4. Rehydrate through an ethanol series as described in Part

A.
9 Chapter 9 Cloning genes by PCR

Chenchik, A., Diachenko, L., Moqadam, F., Tarabykin, V.,

Lukyanov, S. and Siebert, P.D. (1996) Full-length cDNA
cloning and determination of mRNA 5′ and 3′ ends by
amplification of adaptor-ligated cDNA. Biotechniques 21,
526–534.

Flouriot, G., Brand, H. and Gannon, F. (1999)

Identification of differentially expressed 5′-end mRNA
variants by an improved RACE technique (PEETA). Nucleic
Adds Res. 27, 8.

Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid

production of full-length cDNAs from rare transcripts:
amplification using a single gene-specific oligonucleotide
primer. Proc. Natl Acad. Sci. USA 85, 8998–9002.

Knoth, K., Roberds, S., Poteet, C. and Tamkun, M. (1988)

Highly degenerate, inosinecontaining primers specifically
amplify rare cDNA using the polymerase chain reaction.
Nucleic Acids Res. 16, 10932.

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
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239, 1288–1291.

Liu, Y.G. and Whittier, R.F. (1995) Thermal asymmetric

interlaced PCR: automatable amplification and sequencing of
insert end fragments from P1 and YAC clones for chromosome
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Lukyanov, K., Diatchenko, L., Chenchik, A., Nanisetti, A.,

Siebert, P., Usman, N., Matz, M. and Lukyanov, S. (1997)
Construaion of cDNA libraries from small amounts of total
RNA using the suppression PCR effect. Biophys. Biochem.
Res. Commun. 230, 285–288.

Moynihan, T.P., Markham, A.F. and Robinson, P.A. (1996)

Genomic analysis of human multigene families using
chromosome-specific vectorette PCR. Nucleic Acids Res. 24,
4094–4095.

Ochman, H., Gerber, A.S. and Hartl, D.L. (1988) Genetic

applications of an inverse polymerase chain reaction.
Genetics 120, 621–623.

Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner,

D., Powell, S., Anand, R., Smith, J.C. and Markham, A.F.
(1990) A novel, rapid method for the isolation of terminal
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Weber, K.L., Bolander, M.E. and Sarkab, G. (1998) Rapid

acquisition of unknown DNA sequence adjacent to a known
segment by multiplex restriction site PCR. BioTechniques
25, 415–419. Protocol 9.1 5′-RACE Equipment Microcentrifuge
Adjustable heating block or water bath Thermal cycler Gel
electrophoresis tank

Materials and reagents RNA isolation kit (e.g. Qiagen

RNeasy ® minikit) DEPC-treated water Gene-specific primers
1–3 (GSP1–3) Poly-G primer First-strand cDNA synthesis kit
(e.g. ProSTAR™ First-strand RT-PCR kit, Stratagene) or
Reverse transcriptase buffer (AMV 10×RT buffer: 25 mM
Tris-HCl pH 8.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl 2 ). 10 mM
dNTP mix and 2 mM dCTP solution Reverse transcriptase (e.g.
Qiagen) Terminal transferase buffer (10×buffer: 2 M
potassium cacodylate, 250 mM Tris-HCl pH 7.2, 15 mM CoCl 2
, 2.5 μ g ml −1 BSA) Terminal transferase (e.g. New
England Biolabs) Thermostable DNA polymerase and
accompanying buffer (e.g. Pwo DNA polymerase, Boehringer
Mannheim) 0.8% agarose (100 ml: 0.8 g of agarose in 100 ml
of 1×TAE)

Protocol Part A—first-strand cDNA synthesis

1. Add the following to a 1.5 ml microcentrifuge tube

(final reaction volume should be 25 µl):

′ 2.5–5 pmol of GSP1;

′ 1–5 μ q of total RNA;

′ DEPC-treated water up to 12.5 µl.

2. Heat samples to 70°C for 10 min to denature the RNA

followed by chilling on ice.

3. Centrifuge the tubes for 5 s in a microcentrifuge to

collect the entire content.

4. Add the following to each tube:

′ 2.5 µl 10×RT-PCR buffer;

′ 1 µl of 10 mM dNTP;

′ 100–200 units of reverse transcriptase (the amount

required depends on which RT enzyme is used);

′ DEPC-treated water up to 25 µl.

5. Incubate the reaction at 42°C for 1 h followed by

heating to 70°C for 10 min.

6. Sometimes it is advisable to purify the cDNA before

proceeding, but this is not strictly necessary.

Protocol Part B—TdT tailing of the cDNA

1. To each tube add the following:

′ 10 μ l 5×tailing buffer;

′ 2.5 µl of 2 mM dCTP;

′ Water up to 49 µl.

2. Heat the reaction to 94°C for 3 min followed by chilling

on ice.

3. Add 1 µl of TdT to each tube and incubate for 10 min at

37°C followed by heat inactivation at 65°C for 10 min.

Protocol Part C—PCR of tailed cDNA

1. Add the following to a 0.5 or 0.2 μ l PCR tube in a

pre-heated heat block at 94°C:

′ 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

2. Add 0.5 µl of Taq or other thermostable DNA polymerase.

3. Perform a standard PCR amplification.

4. Analyze amplification products on a 0.8% agarose gel.

5. If necessary perform a nested PCR amplification with

GSP3 and the oligodG primer as described in Chapter 5.
Protocol 9.2 Inverse PCR from plant genomic DNA Equipment
Pestle and mortar Adjustable heating block or water bath
Microcentrifuge Thermal cycler Gel electrophoresis tank

Materials and reagents Liquid nitrogen Plant genomic DNA

extraction kit (e.g. Nucleon Phytopure extraction kit,
Amersham Lifesciences) Restriction endonucleases and
accompanying buffer Phenol-chloroform mix (e.g. phenol:
chloroform:iso-amyl alcohol 25: 24:1, GIBCO-BRL) T4-DNA
ligase and accompanying buffer Gene-specific primers and
nested gene-specific primers Thermostable DNA polymerase
and accompanying buffer (e.g. Pwo DNA polymerase,
Boehringer Mannheim) 1% agarose (100 ml: 1g of agarose in
100 ml of 1×TAE)

Protocol Part A—extraction of plant genomic DNA

1. Harvest fresh tissue and snap-freeze using liquid

nitrogen.

2. Grind frozen tissue extensively.

3. Follow manufacturer’s instructions for the subsequent

extraction procedure.

Protocol Part B—restriction endonuclease digestion

1. For each restriction digest use 500 ng of total genomic

DNA in a total volume of 30 µl Check that the genomic DNA
has been digested by gel electrophoresis.

2. Heat inactivate the restriction enzymes according to the

manufacturer’s instructions. If heat stable,
phenol-chloroform-extract

3. Dilute the restriction digest 5-fold in ligation mixture

(ligation buffer, H 2 O, ligase) and incubate at room
temperature for 6–12 hours.

Protocol Part C—first round PCR

1. For each restriction digest use 5 and 10 µl of ligation

reaction for each PCR in a final volume of 50 µl.

2. Use the innermost primers for the first-round PCR

(Figure 9.5; primers 2 and 4).

3. Depending on the primer sequences use high stringency

for annealing to avoid nonspecific priming.
4. Perform a standard PCR using 2 min extension at 72°C and
allow the reaction to proceed for 40 cycles.

5. Run 15 μ l of the PCRs on a 1 % agarose gel.

Protocol Part D—second-round nested PCR

1. Set up three second-round nested PCRs containing 1, 5 µl

and 10 µl of the first-round PCRs.

2. Use nested primers at high stringency (Figure 9.5;

primers 1 and 3).

3. Perform a standard PCR using 1 min extension at 72°C and

allow the reaction to proceed for 35 cycles.

4. Run 20 µl of the PCRs on a 1% agarose gel. Protocol 9.3

Linear PCR and biotin/streptavidin to isolate regions
adjacent to a known DNA sequence Equipment Pestle and
mortar Adjustable heating block or water bath Thermal
cycler Gel electrophoresis tank Magnetic separator (e.g.
Dynal ® magnetic stand)

Materials and reagents Liquid nitrogen Plant genomic DNA

extraction kit (e.g. Nucleon Phytopure extraction kit,
Amersham Lifesciences) Biotinylated gene-specific primer
and nested nonbiotinylated genespecific primers 10 mM dNTP
mix and 2 mM dCTP solution Thermostable DNA polymerase and
accompanying buffer (e.g. Pwo DNA polymerase, Boehringer
Mannheim) Streptavidin coated magnetic beads (e.g. Dynal ®
Dynabeads) Poly-G primer Terminal transferase buffer
(10×buffer: 2 M potassium cacodylate, 250 mM Tris-HCl pH
7.2, 15 mM CoCl 2 , 2.5 μ g ml −1 BSA) Terminal
transferase (e.g. New England Biolabs) 0.8% agarose (100
ml: 0.8 g of agarose in 100 ml of 1×TAE)

Protocol

1. Add the following to a 0.5 or 0.2 μ l PCR tube:

′ 1–3 µl of genomic DNA;

′ 2 µl 10 mM dNTP;

′ 5 μ l of 10×PCR buffer;

′ 50 pmol of a biotinylated primer complementary to the

known DNA sequence priming in the direction of the unknown
DNA sequence;
′ 0.5 μ l of Taq or any other thermostable DNA polymerase
of your choice;

′ water up to 50 µl.

2. Perform a standard PCR amplification for 30–40 cycles.

3. Purify the biotinylated single-stranded DNA using

streptavidin according to the manufacturer’s instructions
(one good supplier is Dynal, who sell magnetic
streptavidin-coated beads) and elute in 25 µl of water.

4. Tail the single-stranded purified DNA as described in

Protocol 9.1.

5. Add the following to a new 0.5 or 0.2 µl PCR tube:

′ 5 μ l of tailed DNA from step 4;

′ 2 µl 10 mM dNTP;

′ 5 µl of 10×PCR buffer;

′ 50 pmol of a nested primer complementary to the known DNA

sequence priming in the direction of the unknown DNA
sequence;

′ 50 pmol of a polyG primer;

′ 0.5 μ l of Taq or any other thermostable DNA polymerase

of your choice;

′ water up to 50 μ l.

6. Perform a standard PCR amplification and analyze the

amplification products on a 0.8% agarose gel.

7. If needed perform a second nested PCR amplification and

compare the outcome with the primary nested reaction in
order to identify the correct amplification product.
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