Polymerase Chain Reaction

1.	Abbreviations and Glossary of Special Terms
2.	Introduction
3.	Principle
4.	Reaction Components
4.1.	DNA Polymerases
4.1.1.	Fidelity
4.1.2.	Mg²⁺ as Cofactor
4.1.3.	Inhibitors
4.2.	Deoxynucleoside Triphosphates (dNTPs)
4.3.	Reaction Buffer
4.4.	Primer
4.4.1.	Melting Temperature
4.4.2.	Concentration
4.4.3.	Synthesis
4.4.4.	Primer Design
4.5.	Template
5.	Equipment and Laboratory Preconditions
6.	Cycling Conditions and Reaction Phases
6.1.	Denaturation Conditions
6.2.	Annealing Conditions
6.3.	Extension Conditions
6.4.	Amplification Phases
7.	Characterization of PCR Products
7.1.	Gel Electrophoresis
7.2.	Restriction Analysis
7.3.	Hybridization
7.4.	Sequencing
8.	Modifications of PCR for Increased Specificity and Sensitivity
8.1.	Increasing Specificity
8.2.	Increasing Sensitivity
9.	Contamination
9.1.	Sources of Contamination
9.2.	PCR Controls
9.3.	Decontamination Methods
9.3.1.	Uracil-N-Glycosylase (UNG)
9.3.2.	UV Light
9.3.3.	Enzymes
9.3.4.	Psoralens and Isopsoralens
10.	Quantitative PCR
10.1.	External Control
10.2.	Internal Control
10.3.	Real-Time Quantification
10.3.1.	Fluorescence Resonance Energy Transfer (FRET)
10.3.2.	Förster Energy Transfer (TaqMan System)
11.	Applications
11.1.	Detection of Pathogenic Agents
11.1.1.	Preconditions
11.1.2.	Extraction of Template DNA
11.1.2.1.	Isolation of Total DNA
11.1.2.2.	Isolation of Pathogen Specific DNA
11.1.3.	Amplification
11.1.4.	Multiplex PCR
11.1.5.	In Situ PCR
11.1.6.	Universal PCR
11.1.7.	PCR Controls for Diagnostic Purposes
11.2.	Detection of Genetic Disorders or Cancerous Cells
11.2.1.	Known Mutations
11.2.1.1.	Amplification Refractory Mutation System (ARMS)
11.2.1.2.	Allele-Specific Oligonucleotides (ASOs)
11.2.1.3.	Introduction of Restriction Sites
11.2.1.4.	Competitive PCR
11.2.1.5.	Primer Extension PCR (PEST)
11.2.2.	Unknown Mutations
11.2.2.1.	Denaturing Gradient Gel Electrophoresis (DGGE)
11.2.2.2.	Single Strand Conformation Polymorphism (SSCP)
11.2.2.3.	Chemical Cleavage of Mismatches (CCM)
11.3.	PCR Analysis in Evolution and Taxonomy
11.3.1.	Restriction Fragment Length Polymorphism
11.3.2.	PCR for Typing Satellites or HLA Genes
11.3.3.	Random Amplified Polymorphic DNA
11.3.4.	Analysis of Fingerprints
11.3.5.	Restrictions and Future Aspects
11.4.	Tissue Typing
11.5.	PCR in Food Analysis
11.6.	PCR to Engineer DNA
11.6.1.	Cloning of PCR Products
11.6.2.	PCR Mutagenesis
11.7.	PCR for Analysis of Gene Expression

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Polymerase Chain Reaction

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Polymerase Chain Reaction

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Polymerase Chain Reaction

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Abstract

General References

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