DNA Manipulation and Analysis

Preface
Contents
Contributors
Chapter 1: Classical Recombinant DNA Cloning
	1 Introduction
		1.1 Historical Perspective
		1.2 Traditional Molecular Cloning Overview
		1.3 Cloning Vectors
		1.4 Restriction Endonucleases
		1.5 Recombinant DNA Production
		1.6 Transformation
		1.7 Selection and Screening
		1.8 Current and Future Applications
	2 Materials
		2.1 Polymerase Chain Reaction (PCR) and Agarose Gel Electrophoresis
		2.2 Restriction Enzyme Digestion
		2.3 Ligation
		2.4 Transformation
		2.5 Selection
		2.6 Screening by PCR
	3 Methods
		3.1 Polymerase Chain Reaction (PCR) and Agarose Gel Electrophoresis
		3.2 Restriction Enzyme Digestion
		3.3 Ligation
		3.4 Transformation
		3.5 Selection
		3.6 Screening by PCR (Colony PCR)
	4 Notes
	References
Chapter 2: A Sequence- and Ligation-Independent Cloning (SLIC) Procedure for the Insertion of Genes into a Plasmid Vector
	1 Introduction
	2 Materials
		2.1 Molecular Biology
		2.2 Gel Analysis
		2.3 Plasmid Preparation
	3 Method
		3.1 PCR Amplification of Insert and Vector
		3.2 Transformation and Sequencing
	4 Notes
	References
Chapter 3: Molecular Cloning Using In Vivo DNA Assembly
	1 Introduction
	2 Materials
		2.1 Polymerase Chain Reaction
		2.2 DNA Gel Electrophoresis and Eliminating Parental DNA
		2.3 Transformation
	3 Methods
		3.1 Primer Design
			3.1.1 Insertion
			3.1.2 Deletion
			3.1.3 Mutagenesis
			3.1.4 Sub-cloning
		3.2 Using PCR for DNA Modification and Amplification
		3.3 Transformation and Colony Selection
		3.4 Performing Complex Procedures
		3.5 Alternative Routes for Linear Fragment Generation: Restriction Enzymes and Synthetic Genes
	4 Notes
	References
Chapter 4: Assembling Multiple Fragments: The Gibson Assembly
	1 Introduction
	2 Materials
		2.1 Enzymes and Primers for Gibson Assembly
		2.2 Generation of the Fragments
		2.3 Bacterial Plasmid Transformation
	3 Methods
		3.1 Design of Overlapping Primers
		3.2 Obtaining the Fragments
		3.3 Assembly
	4 Notes
	References
Chapter 5: TA Cloning Approaches to Cloning DNA with Damaged Ends DNA
	1 Introduction
		1.1 What Is TA Cloning and Why Is It Important?
		1.2 Cloning of Non-PCR-Generated Sequences and End Damage
	2 Materials
		2.1 End Repair
		2.2 Adding the 3′ Adenines
		2.3 Purification
		2.4 Ligation
		2.5 Transformation
		2.6 Screening for Clones
		2.7 Gel Electrophoresis
	3 Methods
		3.1 End Repair
		3.2 Adding the Adenines
		3.3 Purification
		3.4 Ligation
			3.4.1 Using the pGEM-T Easy Vector Systems Kit
			3.4.2 Using the TOPO TA Cloning Kit
		3.5 Transformation
		3.6 Screening for Clones
		3.7 Gel Electrophoresis
	4 Notes
	References
Chapter 6: PCR-Based Assembly of Gene Sequences by Thermodynamically Balanced Inside-Out (TBIO) Gene Synthesis
	1 Introduction
	2 Materials
	3 Methods
		3.1 Primer Design and Synthesis
		3.2 Single-Round TBIO Gene Synthesis
		3.3 Multi-Round TBIO Gene Synthesis
		3.4 Recommended Steps
	4 Notes
	References
Chapter 7: Random Mutagenesis by PCR
	1 Introduction
	2 Materials
		2.1 General Requirements
		2.2 Planning
		2.3 Pilot EP-PCR Experiment
	3 Method
		3.1 Mutagenesis of Homogenous Starting Sequences
		3.2 Mutagenesis of Heterogenous Starting Sequences
	4 Notes
	References
Chapter 8: In Vitro Site Directed Mutagenesis
	1 Introduction
	2 Materials
		2.1 SDM PCR
		2.2 Digestion and Transformation
		2.3 Colony PCR and Sequencing
	3 Methods
		3.1 Selection of Mutations and Design of Primers
		3.2 SDM PCR
		3.3 Digestion of Template DNA and Transformation
		3.4 Colony PCR and Sequencing
	4 Notes
	References
Chapter 9: Xenopus Transgenesis Using the pGateway System
	1 Introduction
		1.1 Transgenesis as a Tool for Reporter Gene Assays
		1.2 The I-SceI Technique and the pGateway System
	2 Materials
		2.1 Plasmid Recombination and Screening
		2.2 Transgenesis in Xenopus and Selection of Transgenic Embryos
	3 Methods
		3.1 Plasmid Recombination and Screening
		3.2 Transgenesis in Xenopus
	4 Notes
	References
Chapter 10: CRISPR/Cas9 Gene Disruption Studies in F0 Xenopus Tadpoles: Understanding Development and Disease in the Frog
	1 Introduction
	2 Materials
		2.1 Target Identification, Oligonucleotide Design and Guide RNA Synthesis
		2.2 Injection Delivery of CRISPR/Cas9 Constructs
		2.3 Analysis of Crispant Tadpoles: Genotyping
		2.4 Analysis of Crispant Tadpoles: Phenotyping
	3 Methods
		3.1 Target Identification and Oligonucleotide Design
		3.2 Guide RNA Synthesis
		3.3 Injection of CRISPR/Cas9 Constructs into Xenopus Embryos
		3.4 Genotyping Embryos
		3.5 Phenotyping Mutant Embryos
	4 Notes
	References
Chapter 11: A CRISPR/Cas-Based Method for Precise DNA Integration in Xenopus laevis Oocytes Followed by Intracytoplasmic Sperm...
	1 Introduction
	2 Materials
		2.1 BAC Cloning
		2.2 Long Single Stranded DNA Synthesis
			2.2.1 Gibson Assembly
			2.2.2 Long Single Stranded DNA Synthesis
		2.3 Oocyte Preparation and Culture
		2.4 CRISPR Injection
		2.5 Sperm Nuclei Preparation
		2.6 Intracytoplasmic Sperm Injection (ICSI)
	3 Methods
		3.1 Designing sgRNA and lssDNA Inserts
		3.2 BAC Cloning
		3.3 Long Single Stranded DNA (lssDNA) Insert Synthesis
			3.3.1 Gibson Assembly Cloning
			3.3.2 Single Stranded DNA Synthesis
		3.4 Oocyte Preparation
		3.5 CRISPR/Cas9 Injection in Oocytes
		3.6 Oocyte Culture and Maturation
		3.7 Sperm Nuclei Preparation
		3.8 ICSI Fertilization in Oocytes
	4 Notes
	References
Chapter 12: A Lambda-Exonuclease SELEX Method for Generating Aptamers to Bacterial Targets
	1 Introduction
	2 Materials
		2.1 Preparation of Bacterial Target
		2.2 SELEX Round One: Binding
		2.3 SELEX Round One: Purifying Bound DNA
		2.4 SELEX Round One: Amplification and Regeneration of ssDNA
		2.5 Further SELEX Rounds
		2.6 Cloning and Sequencing of Aptamer Candidates
		2.7 Analyzing and Ordering Aptamer Candidates
	3 Methods
		3.1 Preparation of Bacterial Target
		3.2 SELEX Round One: Binding
		3.3 SELEX Round One: Purifying Bound DNA
		3.4 SELEX Round One: Amplification and Regeneration of ssDNA
		3.5 Further SELEX Rounds
		3.6 Cloning and Sequencing of Aptamer Candidates
		3.7 Analyzing and Ordering Aptamer Candidates
	4 Notes
	References
Chapter 13: Generation of Functional-RNA Arrays by In Vitro Transcription and In Situ RNA Capture for the Detection of RNA-RNA...
	1 Introduction
	2 Materials
		2.1 In Vitro Transcription Template Design and Synthesis
		2.2 Generation of the DNA In Vitro Transcription Template Array
		2.3 Generation of the Functional-RNA Array
		2.4 Application of Functional-RNA Arrays to the Evaluation of RNA-RNA Binding Specificity
	3 Methods
		3.1 In Vitro Transcription Template Design and Synthesis
		3.2 Generation of the DNA In Vitro Transcription Template Array
		3.3 Generation of the Functional-RNA Array
		3.4 Application of Functional-RNA Arrays to the Evaluation of RNA-RNA Binding Specificity
	4 Notes
	References
Chapter 14: Chemical Synthesis of Oligonucelotide Sequences: Phosphoramidite Chemistry
	1 Introduction
	2 Materials
		2.1 Standard Synthesis (See Note 1)
		2.2 Oligonucleotide Purification Using Reverse-Phase HPLC (RP-HPLC)
		2.3 Final Desalting of the Oligonucleotide Solution Using RP-HPLC
	3 Methods
		3.1 Automated Oligonucleotide Synthesis
			3.1.1 Cleavage and Deprotection
		3.2 Oligonucleotide Purification Using Reverse Phase-HPLC (RP-HPLC) (See Note 18)
		3.3 Final Desalting of the Oligonucleotide Solution Using RP-HPLC
	4 Notes
Chapter 15: Low Throughput Direct Cycle Sequencing of Polymerase Chain Reaction (PCR) Products
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Kits, Reagents, and Consumables
	3 Methods
		3.1 PCR Product Purification and Clean up
		3.2 Specific PCR Product
		3.3 Excision Gel Protocol
		3.4 PCR Product Quality and Quantity Assessment
			3.4.1 Measurement of Concentration with Nanodrop 2000
			3.4.2 Agarose Gel Electrophoresis
		3.5 Cycle Sequencing
			3.5.1 Considerations for Sequencing Primer Selection and Design
			3.5.2 Cycle Sequencing Reaction
		3.6 Cycle Sequencing Product Purification
		3.7 Sample Preparation
		3.8 Running Sequencing on the Capillary Electrophoresis System (Seqstudio)
		3.9 DNA Sequence Viewing and Analysis
		3.10 Troubleshooting
			3.10.1 Unincorporated Dye Blob
			3.10.2 Poor Start Followed by a Weak Signal
			3.10.3 Overlapping Peaks
			3.10.4 Failed Sequence
			3.10.5 Secondary Structure
	4 Notes
	References
Chapter 16: Nanopore Sequencing for Mixed Samples
	1 Introduction
	2 Materials
		2.1 Consumables
		2.2 Reagents
		2.3 Equipment
	3 Method
		3.1 Setting Up
		3.2 Post-PCR Quality Control
		3.3 Sample Dilution, End Repair and dA-Tailing
		3.4 Barcode Ligation
		3.5 Barcode Clean-Up
		3.6 Sequencing Adapter Ligation and Clean-Up
		3.7 Sequencing Preparation
			3.7.1 Library Preparation and Loading
	4 Notes
	References
Chapter 17: Ethics, Legality, and Safety for Geneticists
	1 Introduction
	2 Research Integrity
	3 Research Governance
		3.1 Health and Safety (Physical, Chemical, Biological)
		3.2 Genetically Modified Organism Legislation/Policy
		3.3 Ethics Policies
	4 Independent Review
	5 Conclusion
	References
Index