PCR: Methods and Protocols (Methods in Molecular Biology, 2967)

✍ Scribed by Lucília Domingues (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 255
• Edition: 2nd ed. 2023
• Category: Library

✦ Synopsis

This second volume focuses on PCR methods and PCR application specificities to the biotechnology and bioengineering field. New and updated chapters detail real-time PCR protocols, synthetic biology applications, pathogen detection, microfluidics, digital, multiplex detection recent advances. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, PCR: Methods and Protocols, Second Edition aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Digital PCR: A Partitioning-Based Application for Detection and Surveillance of SARS-CoV-2 from Sewage Samples
1 Introduction
2 Materials
2.1 Wastewater Sample Collection
2.2 Filtration, Centrifugation, and Precipitation
2.3 RNA Extraction from Concentrated Pellet
2.4 Primer and Probe Design for Digital PCR
2.5 Digital PCR Mix Preparation
2.6 Software Setup and Thermal Cycling Conditions
3 Methods
3.1 Wastewater Sample Collection
3.2 Filtration, Centrifugation, and Precipitation
3.3 RNA Extraction from Concentrated Pellet
3.4 Primer and Probe for Digital PCR
3.5 Digital PCR Mix Preparation for Wastewater Samples
3.6 Software Setup and Thermal Cycling Conditions
3.7 Result Interpretation and Data Analysis
4 Notes
References
Chapter 2: Digital PCR: A Tool to Authenticate Herbal Products and Spices
1 Introduction
2 Materials
2.1 Preparation of Blended Formulations
2.2 DNA Extraction, Quantification, and Dilution
2.3 Primer Dilution
2.4 Preparation of Reaction Mixture
3 Methods
3.1 Preparation of Blended Formulations
3.2 DNA Extraction, Quantification, and Dilution
3.3 Digital PCR Setup
3.4 PCR Thermal Cycle and Run Set Up in QIAcuity Digital PCR System
3.5 Data Collection and Analysis
4 Notes
References
Chapter 3: Emulsion Polymerase Chain Reaction Coupled with Denaturing Gradient Gel Electrophoresis for Microbial Diversity Stu...
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 PCR Amplification
2.3 Droplet Generator
2.4 Emulsion PCR
2.5 Polyacrylamide Gel-DGGE
3 Methods
3.1 DNA Extraction
3.2 Emulsion PCR Amplification
3.3 Emulsion PCR-DGGE
3.3.1 Preparation of Polyacrylamide Gel for DGGE Analysis
3.3.2 Assembling the D-Code (Gel Running) System
3.3.3 Loading the Gel
3.3.4 Staining and Imaging the Gel
4 Notes
References
Chapter 4: Real-Time PCR High-Resolution Melting Assays for the Detection of Foodborne Pathogens
1 Introduction
2 Materials
2.1 DNA Purification
2.2 PCR
3 Methods
3.1 Primer Design
3.2 Real-Time PCR
4 Notes
References
Chapter 5: High-Throughput Real-Time qPCR and High-Resolution Melting (HRM) Assay for Fungal Detection in Plant Matrices
1 Introduction
2 Materials
3 Methods
3.1 qPCR
3.1.1 Experimental Design of qPCR
3.1.2 Standard Curve Design
3.1.3 Quantitative PCR Assay
3.2 High-Resolution Melting
4 Notes
References
Chapter 6: Multiplex Real-Time PCR for the Detection of Shiga Toxin-Producing Escherichia coli in Foods
1 Introduction
2 Materials
2.1 Media (See Note 1)
2.2 Chemicals
2.3 Equipment
3 Methods
3.1 Bacterial Enrichment
3.2 DNA extraction (see Note 3)
3.3 DNA Quantification
3.4 STEC Multiplex qPCR Assay
4 Notes
References
Chapter 7: DNA Isolation from Cocoa-Derived Products and Cocoa Authentication by TaqMan Real-Time PCR
1 Introduction
2 Materials
2.1 General Supplies
2.2 DNA Extraction
2.3 Real-Time PCR
3 Methods
3.1 DNA Extraction
3.2 TaqMan Real-Time PCR
3.3 Thermocycling
3.4 Program and Run
3.5 Data Collection and Analysis
4 Notes
References
Chapter 8: Quantitative Real-Time PCR for the Detection of Allergenic Species in Foods
1 Introduction
2 Materials
2.1 Bioinformatic Tools
2.2 Reagents
2.3 Equipment
3 Methods
3.1 In Silico Analysis
3.2 Preparation of Model Foods
3.3 DNA Extraction
3.4 Determination of DNA Yield and Purity
3.5 Qualitative PCR
3.6 DNA Sequencing
3.7 Real-Time PCR with Hydrolysis Probe
3.8 Real-Time PCR Validation
4 Notes
References
Chapter 9: Accurate Absolute Quantification of Bacterial Populations in Mixed Cultures by qPCR
1 Introduction
2 Materials
2.1 Culture Media and Bacterial Species
2.2 Genomic DNA Extraction
2.3 qPCR Material
3 Methods
3.1 Preparation of Bacterial Cultures for gDNA Calibration Curves
3.2 gDNA Extraction Using a Commercial Kit
3.3 qPCR Efficiency
3.4 qPCR Calibration Curve Construction for Each Target Species
3.5 Absolute Quantification of Bacterial Species Concentration in Polymicrobial Samples
4 Notes
References
Chapter 10: Real-Time PCR Method for Assessment of ParA-Mediated Recombination Efficiency in Minicircle Production
1 Introduction
2 Materials
2.1 Recombination Efficiency Determination
2.2 Agarose Gel Electrophoresis
3 Methods
3.1 Primer Design
3.2 Calibration Curves Preparation
3.3 Determination of Recombination Efficiency
4 Notes
References
Chapter 11: Gene Expression Quantification from Pathogenic Bacterial Biofilms by Quantitative PCR
1 Introduction
2 Materials
2.1 Sample Collection and RNA Isolation
2.2 DNase Treatment
2.3 RNA Quality Assessment
2.4 Complementary DNA Synthesis
2.5 Quantitative PCR
3 Methods
3.1 Sample Collection
3.2 RNA Isolation
3.3 Genomic DNA Degradation/Removal
3.4 RNA Quality Assessment
3.4.1 RNA Quantity and Purity
3.4.2 RNA Integrity
3.5 Complementary DNA Synthesis
3.6 Quantitative PCR Run
4 Notes
References
Chapter 12: A Real-Time Quantitative PCR Protocol for the Quantification of Plasmid Copy Number in Lactococcus lactis
1 Introduction
2 Materials
2.1 L. lactis PCN Determination by Real-Time Quantitative PCR
3 Methods
3.1 L. lactis PCN Determination by Real-Time Quantitative PCR
4 Notes
References
Chapter 13: Improved PCR by the Use of Disruptors, a New Class of Oligonucleotide Reagents
1 Introduction
2 Materials
2.1 PCR
2.2 Agarose Gel Electrophoresis
3 Methods
3.1 Secondary Structure Prediction of Amplicon Sequence
3.2 Disruptor Design
3.3 PCR
3.4 Result Analysis
4 Notes
References
Chapter 14: Mitochondrial DNA D-Loop Amplification and Sequencing for Species Differentiation in Milk
1 Introduction
2 Materials
2.1 Equipment
2.2 Supplies and Reagents
3 Methods
3.1 Total DNA Extraction in Milk
3.2 PCR Amplification of Mitochondrial D-Loop DNA
3.3 Validation
4 Notes
References
Chapter 15: Long-Range Polymerase Chain Reaction
1 Introduction
2 Materials
2.1 PCR
2.2 Agarose Gel Electrophoresis
3 Methods
3.1 Primer Design
3.1.1 Obtaining Reference Sequence
3.1.2 Obtaining Specific Primer Sequences (Fig. 2 (See Note 12))
3.2 PCR
3.3 Agarose Gel Electrophoresis (See Note 19)
4 Notes
References
Chapter 16: Megaprimer-Based PCR to Synthesize Fusion Genes for Cloning
1 Introduction
2 Materials
2.1 Equipment
2.2 Supplies and Reagents
3 Methods
3.1 Primer Design
3.2 Double-Step Primer Extension PCR: Case Study 1
3.3 Standard PCR: Case Study 2
3.4 Vector Cloning and Confirmation of the Fusion Genes´ Sequence
4 Notes
References
Chapter 17: Bacteria and Yeast Colony PCR
1 Introduction
1.1 DNA Release
1.2 Future Developments
2 Materials
2.1 E. coli Colony PCR
2.2 S. cerevisiae Colony PCR Using a Microwave Oven
2.3 PCR Using S. cerevisiae LiAc Permeabilized Cells
3 Methods
3.1 E. coli Colony PCR
3.2 S. cerevisiae Colony PCR Using a Microwave Oven
3.3 PCR Using S. cerevisiae LiAc Permeabilized Cells
4 Notes
References
Chapter 18: Inverse PCR for Site-Directed Mutagenesis
1 Introduction
2 Materials
2.1 iPCR Mutant Amplification
2.2 Agarose Gel Electrophoresis
2.3 Template Removal and Product Recircularization
2.4 Preparation of Chemically Competent E. coli XL1-Blue
2.5 Transformation
2.6 Mutant Confirmation
3 Methods
3.1 iPCR Mutant Amplification
3.2 Template Removal and Product Recircularization
3.3 Transformation and Mutant Confirmation
4 Notes
References
Chapter 19: Optimized Design of Degenerate Primers for PCR Based on DNA or Protein Sequence Comparisons
1 Introduction
2 Materials
2.1 Sequence Manipulation and Primer Design
2.2 PCR, Gel Electrophoresis, and Image Acquisition
2.3 Reagents
3 Methods
3.1 Primer Design from Protein Family Alignment: Reduction of 5′-End Complexity
3.2 Primer Design from DNA Alignment (I): Reduction of Specificity by Degeneration of 5′-End
3.3 Primer Design from DNA Alignment (II): Reduction of Degeneration by Sequence Clustering
3.4 Protocol for Protein or DNA Sequence Analysis
3.5 PCR
3.6 Thermocycling
3.7 Gel Analysis
4 Notes
Bibliography
Index