Principles and Technical Aspects of PCR Amplification
by Elizabeth van Pelt-Verkuil (Author), Alexander van Belkum (Author), John P. Hays (Author)
Product Details
* Hardcover: 339 pages
* Publisher: Springer; 1 edition (April 2008)
* Language: English
* ISBN-10: 1402062400
Book Description
Kary Mullis was awarded a Nobel Prize for inventing the PCR technique more than 15 years ago in 1993. Since its "discovery", multiple adaptations and variations of the standard PCR technique have been described, with many of these adaptations and variations currently being used in clinical, diagnostic and academic laboratories across the world. Further, these techniques are being applied at the diagnostic level (e.g. as high throughput testing methodologies to detect minimum residual disease, the presence/absence of specific pathogens etc), as well as to increase our understanding of fundamental disease processes.
Frequently, PCR technicians and specialists limit their understanding of PCR to one particular methodology. However, this approach limits their appreciation of the range of versatile PCR techniques currently available, techniques that may be applicable and indeed more suitable to their own laboratory situation.
This manual aims to provide the reader with a guide to the standard PCR technique and its many available modifications, with particular emphasis on the role of PCR techniques in the diagnostic laboratory (the central theme of this manual). Further, many important technical issues have been addressed, including types of PCR template material, PCR optimization, the analysis of PCR products, quality control and quality assurance, variants and adaptations of the standard PCR protocol, quantitative PCR and in situ PCR. The reader of this manual will be excellently informed about the fundamental principles of PCR and the true potential of PCR within clinical laboratory practice.
Contents
Foreword
Chapter 1 The Polymerase Chain Reaction
1.1 An Overview of the PCR Process
1.2 Before PCR and Beyond
Chapter 2 A Brief Comparison Between In Vivo DNA Replication
and In Vitro PCR Amplifi cation
2.1 Nucleic Acid Targets
2.1.1 DNA
2.1.2 RNA
2.2 Target DNA Strand Separation and Primer Annealing
2.3 DNA Dependent DNA Polymerase and Oligonucleotide
Primers
2.4 Deoxyribonucleotides and Additional Factors
Chapter 3 The PCR in Practice
3.1 Brief Overview of PCR Requirements
3.1.1 The PCR Reaction Mix
3.1.2 The PCR Thermocycling Regime
3.1.3 Analysis of PCR Amplifi cation Products
3.1.4 Miscellaneous Considerations
Chapter 4 The Different Types and Varieties of Nucleic Acid
Target Molecules
4.1 General Features
4.2 A Brief Description of In Vivo DNA and RNA Targets
4.3 DNA Samples
4.3.1 DNA Isolation Procedures
4.3.2 Comments on Nucleic Acids in Specifi c
Sample Types
4.4 RNA Samples
4.4.1 Working Free of RNase Contamination
4.4.2 RNA Isolation for RT-PCR
4.5 Reverse Transcription and RT-PCR
4.5.1 cDNA Synthesis
4.5.2 cDNA Synthesis Using RACE
4.5.3 RNA Extraction and cDNA
Synthesis Controls
Chapter 5 PCR Primers
5.1 PCR Primer Design and Quality Requirements
5.1.1 Different Primer Species
5.2 Primer Hybridisation (Annealing)
5.3 Thermodynamic Approach of Tm Calculations
5.4 Primer Synthesis
5.5 Non-radioactive Primer Labelling
5.6 The Effect of Mismatches Between PCR
Primer and Target
5.7 Primer Concentration
Chapter 6 Deoxynucleotide Triphosphates and Buffer Components
6.1 Factors Affecting the Choice of dNTP Concentration
6.2 Modifi ed dNTPs and Their Applications
6.3 The PCR Buffer
6.3.1 Monovalent Ions
6.3.2 Magnesium Ions
Chapter 7 Taq and Other Thermostable DNA Polymerases
7.1 The Advantages and Disadvantages of Taq over
Klenow Fragment DNA Polymerase
7.2 Misincorporation of Nucleotides and Fidelity
of DNA Synthesis by Taq Polymerase
7.3 Taq DNA Polymerase and Its Modifi cations
7.4 Taq Polymerase Unit Defi nition and Working
Concentrations
7.5 Other Thermostable Polymerases
and Their Applications
7.6 Mixtures of Thermostable Polymerases
Chapter 8 Important Considerations for Typical, Quantitative
and Real-Time PCR Protocols
8.1 The Typical PCR Amplifi cation Protocol
8.1.1 Denaturation (Melting) of the Template DNA
8.1.2 Annealing (Hybridisation) of PCR Primers
8.1.3 Calculating the Primer Annealing
Temperature (Tm)
8.1.4 DNA Chain Extension/Elongation
8.1.5 PCR Cycle Number
8.1.6 The “Plateau Phase” and Final Stages of PCR
Thermocycling
8.1.7 PCR Sensitivity
8.2 Quantitative PCR Protocols
8.2.1 Quantitative PCR Controls
8.3 Real-Time PCR Protocols
8.4 RNA Extraction and Treatment
Chapter 9 Analysis of PCR Amplifi cation Products
9.1 Visualizing PCR Amplifi cation Products
9.1.1 Intercalating Chemical Dyes and Silver Ions
9.1.2 Fluorescent or Hapten Labelled Amplimers
9.2 Post-PCR Electrophoretic Analysis of Amplimers
9.2.1 Gel Electrophoresis Methodologies
9.2.2 Probe Hybridisation Methodologies
9.3 Real-Time Analysis of PCR Amplimers
9.3.1 In vitro Analysis Using Intercalating
Chemical Dyes
9.3.2 FRET Quenching Assays
9.3.3 TaqMan Probes
9.3.4 FRET Enhancement Reactions
9.4 Nucleic Acid Sequencing
9.4.1 DNA Sequencing Using Non-thermostable
DNA Polymerases
9.4.2 PCR Sequencing Using Thermostable
DNA Polymerases
9.4.3 The Fidelity of PCR Sequencing Reactions
Chapter 10 Ensuring PCR Quality – Laboratory Organisation, PCR
Optimization and Controls
10.1 The Primary Level of Quality Control – Laboratory
Organization and the Prevention of PCR
Contamination
10.1.1 Sources and Routes of Contamination
10.1.2 PCR Contamination Issues Within
Individual PCR Laboratories
10.1.3 Detecting and Preventing PCR
Contamination
10.2 The Secondary Level of PCR Quality Control – PCR
Design and Optimization
10.2.1 Extrinsic and Intrinsic Factors
10.2.2 The Developmental Steps Needed
to Achieve High Quality PCR Results
10.2.3 The Use of Positive and Negative Controls
in PCR Quality
10.2.4 Causes and Solutions for False Positive
and False Negative PCR Results
10.3 Quality Considerations Specifi c for RT-PCR
Methodologies
10.3.1 Problems Likely to Cause False Positive
Results in RT-PCR Assays
10.3.2 Problems Likely to Cause False Negative
Results in RT-PCR Assays
Chapter 11 Ensuring PCR Quality – Quality Criteria and Quality
Assurance
11.1 Quality Control Criteria and PCR
11.1.1 Sensitivity and Diagnostic Sensitivity
11.1.2 Specifi city and Diagnostic Specifi city
11.1.3 Reference and Threshold Values
11.1.4 The Predictive Value
11.1.5 Effi ciency
11.1.6 Error and Accuracy
11.1.7 Precision and Correctness
11.1.8 Defi ning the Analytical or Quantifi cation
Range and Sensitivity
11.1.9 Recovery, Reproducibility and Quality
Assurance
11.2 Quality Assurance and Multicenter Studies
Chapter 12 Variants and Adaptations of the Standard PCR Protocol
12.1 Generating Labelled PCR Amplimers for PCR
Product Visualization, DNA Probes and Cloning
12.2 Two-Step PCR Protocol
12.3 Booster PCR
12.4 Hot-Start and Time-Release PCR Protocols
12.5 Inverse PCR
12.6 Asymmetric PCR
12.7 PCR Mediated DNA Sequencing Strategies
12.7.1 Generating Single-Stranded DNA
for Sanger Sequencing Reactions
12.7.2 Classical Sanger Sequencing
of Single-Stranded PCR Products
12.7.3 Direct PCR Sequencing
12.7.4 Four-Tube Cycle Sequencing
12.7.5 One-Tube Cycle Sequencing
12.7.6 Diffi cult to Sequence Templates
12.8 Touchdown and Touch-Up PCR
12.9 Multiplex PCR
12.10 PCR Using Degenerate Primers
12.11 Repeat and Inter-repeat PCR
12.11.1 Repeat PCR
12.11.2 Inter-repeat PCR and Random Amplifi cation
of Polymorphic DNA (RAPD)
12.12 AFLP Fingerprinting
12.13 Base Excision Sequence Scanning (BESS-T-Scan)
for Mutation Detection
12.14 Differential Display RT-PCR (DD-PCR)
12.15 The Protein Truncation Test (PTT)
12.16 Methylation Specifi c PCR and PCR in the Detection
of Mutagens
12.17 Breakpoint PCR
12.18 Site Directed Mutagenesis by PCR
12.19 PCR Amplimers for Cloning and Expression
12.20 SAGE
12.21 PCR Inhibition by DNA Specifi c Antibiotics
and Mutagens
Chapter 13 In Situ PCR Amplifi cation (ISA) – Major Considerations,
Sample Processing and Applications
13.1 Tissue Processing – Nucleic Acid Fixation/Extraction
13.1.1 Fixation
13.1.2 Type of Nucleic Acid
13.1.3 Detrimental Effects of Various Fixatives
on Nucleic Acids
13.1.4 Effects of Tissue Processing Steps
(Decalcifi cation, Dehydration, Intermedium
Application, Embedding) and Storage
of Paraffi n Blocks
13.1.5 Effects of Histological and Histochemical
Staining
13.2 Differences in Approach for ISH, ISA and
Standard PCR
13.2.1 Different Types of Tissue Preparations .
13.2.2 DNA and RT-PCR on Paraffi n-Embedded
Tissue Sections
13.2.3 Improvement of PCR Effi ciency Using
Fixed Tissue Sections
13.3 An Introduction to In Situ Amplifi cation (ISA)
13.4 Considerations in the Development of ISA Protocols
13.4.1 IS-PCR or PCR-ISH
13.4.2 Diffusion of Nucleic Acids
13.4.3 The Correct Fixative
13.4.4 Damage Caused by Paraffi n Embedding
13.4.5 Detachment of Cells and Tissue Sections
13.4.6 Specimen Proteolysis
13.4.7 Acetylation and Other Forms of Tissue
Section Pre-treatment
13.4.8 Pre-treatment of Preparations for IS-PCR
13.4.9 Testing for Loss of Amplimers Due
to Leakage from Their Site of Production
13.4.10 Miscellaneous IS-PCR Considerations
13.4.11 Mispriming
13.4.12 Primer Independent Non-specifi c
DNA Synthesis
13.4.13 Evaporation of Reactants During IS-PCR/
Wet Hot Start Procedure
13.4.14 Cell Thickness and ISA
13.4.15 Choosing a Hybridisation Control
for Testing Amplimer Specifi city
13.4.16 Choice of the PCR Processor
13.4.17 Choice of the Final Detection Method
13.5 ISA Optimisation
13.6 ISA Controls
Index
