Second Edition � edited by
Thomas Weissensteiner et al

• Provides up-to-date, essential information on PCR technology and applications
• Includes a selection of widely used procedures and approaches for qualitative as well as quantitative queries
• Explains parameters governing PCR reaction outcomes
• Presents protocols in easy-to-follow, step-by-step presentations
• Covers primer design, enzyme choices, precautions to avoid false positives, and more

PCR Technology: Current Innovations, Second Edition is a fundamental reference for scientists new to PCR technology and a source of up-to-date applications for those familiar with the method. It provides theoretical considerations, discussions, and a selection of state-of-the-art techniques for mutation studies, clinical diagnosis, and the detection of food-borne pathogens.

The book begins with discussions of the preparation of PCR experiments, followed by examples of analytical PCR divided into qualitative and quantitative applications. The final section explores preparative methods addressing DNA generation for further analysis and in vitro evolution.

Featuring detailed presentations of PCR protocols, this volume contains critical information for practitioners in a wide variety of fields, including forensics, molecular biology research, clinical DNA diagnostics, botany, and paleontology. It provides the essential tools for developing innovative approaches in this leading technology of the genomics revolution.

Contents

1. General Considerations
   1. Template Isolation and Preparation
      • Extraction and Amplification of Ancient Data
      • Long PCR from Damaged DNA
      • Quantitative mRNA Analysis in Small Cell Samples by RT-PCR and Flow Cytometry
      • Microdissection of Single or Small Numbers of Cells for Analyses of DNA and RNA by PCR
   2. PCR Optimization
      • Pre-PCR Processing Strategies
      • Novel PCR-Enhancing Compounds and their Modes of Action
      • Optimizing PCR with the Aid of Experimental Design
   3. Labeling
      • Economic Fluorescent Labeling of PCR-Products for Microsatellite- and Single-Stranded-Conformation-Polymorphism (SSCP) Analysis
   4. Cloning
      • Universal Restriction Site-Free Cloning Method Using Chimeric Primers

2. Analytical Applications
   5. Detection of Nucleic Acid Variation
      • High-Throughput Genotyping and the PCR
      • High Throughput Genetic Analysis Through Multiplexed PCR and Multi-Capillary Electrophoresis
      • The Good Assay: A Purification Free Assay for Genotyping by Maldi Mass Spectrometry
      • Primer Design for Large Scale Multiplex PCR and Arrayed Primer Extension (APEX)
      • Surface Plasmon Resonance Based Biosensor Technology for Real-Time Detection of PCR Products
      • Fluorescent Amplified Fragment Length Polymorphism (FAFLP) Genotyping
      • Global Analysis of DNA Allelic Variations (GADAV) by Specific Enrichment of Mismatches and Selective Amplification of Heterohybrids
      • Quantitative DNA-Methylation Analysis by the Bisulfite Conversion Method
   6. Sequencing
      • Pyrosequencing Technology
      • Octamer-Primed Cycle Sequencing
   7. In Situ Analysis
      • In Situ Amplification of cDNA
      • Detection of a Single Copy Sequence on Human by Cyclic Prins
   8. Quantitative Analysis
      • Meaningful Quantification of mRNA Using Real-Time PCR
      • Absolute Quantification of Specific Nucleic Acids by (RT)-PCR Using a Nonlinear Mathematical Model for Data Analysis
      • Single-Molecule PCR - Basic Protocols and Applications
      • Gene Expression and Discovery: Application of a Genome-Wide Approach
      • The Detection of Differential Gene Expression Changes Using SAGE: (Serial Analysis Of Gene Expression)
      • Applications of Differential Display in Infectious Diseases
      • Restriction Endonucleolytic Analysis of Differentially Expressed Sequences: READS
      • Suppression Subtractive Hybridization PCR

3. Preparative Applications
   9. Amplification of Unknown Sequences
      • PCR-Based Techniques for Cloning an Unknown DNA Fragment Adjacent to a Known Integrant
      • Chromosome Walking By Inverse PCR
      • Long Universal Fast Walking
      • Cloning of Unknown Virus Sequences By DNase Treatment and Sequence-Independent Single Primer Amplification
      • Whole Genome Amplification from Single Cells and Minute DNA Samples
      • Primer Extension Pre-Amplification (PEP) of Single Cells: Efficiency and Bias
   10. In Vitro Evolution
       • In Vitro Evolution Through PCR-Mediated Mutagenesis and Recombination

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