Polymerase Chain Reaction

Abstract The article contains sections titled: 1 Introduction 2 Principle 3 Reaction Components 3.1 DNA Polymerases 3.1.1 Fidelity 3.1.2 Mg 2+ as Cofactor 3.1.3 Inhibitors 3.2 Deoxynucleoside Triphosphates (dNTPs) 3.3 Reaction Buffer 3.4 Ready‐to‐Use Master Mixes 3.5 Primer 3.5.1 Melting Temperature 3.5.2 Concentration 3.5.3 Primer Design 3.6 Template 4 Equipment and Laboratory Preconditions 5 Cycling Conditions and Reaction Phases 5.1 Denaturation Conditions 5.2 Annealing Conditions 5.3 Extension Conditions 5.4 Amplification Phases 6 Characterization of PCR Products 6.1 Gel Electrophoresis 6.2 Restriction Analysis 6.3 Hybridization 6.4 Sequencing 7 Modifications of PCR for Increased Specificity and Sensitivity 7.1 Increasing Specificity 7.2 Increasing Sensitivity 8 Contamination 8.1 Sources of Contamination 8.2 PCR Controls 8.3 Decontamination Methods 8.3.1 Uracil‐ N ‐Glycosylase (UNG) 8.3.2 UV Light 8.3.3 Enzymes 8.3.4 Psoralens and Isopsoralens 9 Quantitative PCR 9.1 External Control 9.2 Internal Control 9.3 Real‐Time PCR (qPCR) 9.3.1 Fluorescent Dyes 9.3.2 Fluorophore‐labeled Probes 9.4 Digital PCR (dPCR) 10 Applications 10.1 Detection of Pathogenic Agents 10.1.1 Preconditions 10.1.2 Extraction of Template DNA 10.1.2.1 Isolation of Total DNA