PCR

PCR

Second Edition

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

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New York, N Y 10016

In UK: 4 Park Square, Milton Park

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© 2006 by Taylor & Francis Group

First published 2000; Second edition published 2006

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

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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

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

vi Contents

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

Contents vii

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

viii Contents

8-MOP 8-methoxypsoralen

8-oxo-dGTP 8-oxo-2′deoxyguanosine

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

BAC bacterial artificial

chromosome

BCIP 5-bromo, 4-chloro, 3-

indolyl phosphate

CAPS cleaved amplified

polymorphic sequence

analysis

CcdB control of cell death

Ct threshold cycle

CCD charge coupled device

cDNA complementary DNA

CT comparative threshold

DHPLC denaturing-high￾performance liquid

chromatography

DIG digoxigenin

DIG-dUTP digoxigenin-11-2′-

deoxyuridine-5′-

triphosphate

DOP-PCR degenerate oligonucleotide

primed-PCR

dPTP 6-(2-deoxy-β-D￾ribofuranosyl)-3,4-dihydro￾8H-pyrimido-[4,5-C][1,2]

oxazin-7-one

ELISA enzyme linked

immunosorbent assay

EST expressed sequence tag

FAM 6-carboxyfluorescein

FDD fluorescent differential

display

FRET fluorescence resonance

energy transfer

FS fluorescent sequencing

GAPDH glyceraldehyde-3-phosphate

dehydrogenase

GAWTS gene amplification with

transcript sequencing

GM genetically modified

HEX 4,7,2′,4′,5′,7′-hexachloro-6-

carboxyfluorescein

HRP horseradish peroxidase

IPCR inverse polymerase chain

reaction

LCR ligase chain reaction

LIC ligation-independent

cloning

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

PCR-VNTRs PCR highly polymorphic

variable number tandem

repeats

PEETA Primer extension,

Electrophoresis, Elution,

Tailing, Amplification

PMBC peripheral blood

mononuclear cells

PMT photomultiplier tube

Abbreviations

PNA peptide nucleic acid

PORA NADPH:

protochlorophyllide

oxidoreductase

RACE rapid amplification of cDNA

ends

RACHITT random chimeragenesis on

transient templates

RAPD random amplified

polymorphic DNA

RAWIT RNA amplification with in

vitro translation

RAWTS RNA amplification with

transcript sequencing

RFLP restriction fragment length

polymorphism

RISC RNA-induced silencing

complex

RNAi RNA interference

RT reverse transcriptase

SDS sodium dodecyl sulfate

siRNAs small interfering RNAs

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

TAMRA 6-carboxytetramethyl￾rhodamine

TBR tris (2,2′-bipyridine)

ruthenium (II) chelate

TCA trichloroacetic acid

TdT terminal deoxynucleotidyl￾transferase

TEMED N,N,N′,N′-

tetramethylenediamine

TET 4,7,2′,7-tetrachloro-6-

carboxy fluorescein

TK thymidine kinase

Tm melting temperature

TNF tumor necrosis factor

TOPO ligation topoisomerase-mediated

ligation

Tp optimized annealing

temperature

UNG uracil N-glycosylase

USE unique site elimination

VNTR variable number tandem

repeats

YAC yeast artificial

chromosome

x Abbreviations

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’

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￾1

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).

2 PCR

Primer

DNA

polymerase

Template

Synthesis of new DNA strand

5' 3'

3' 5'

5'

dNTPs

T G

T

T

C

C

A C

A

G

G A

T

T

T

G

G

A G G

A

A C

C C C

3'

3'

5'

5'

3'

A AA

A A

G G

G

T T

T TT C C

C

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.