Synthetic DNA: Methods and Protocols (Methods in Molecular Biology, 1472)

✍ Scribed by Randall A. Hughes (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 250
• Category: Library

✦ Synopsis

This volume presents state-of-the art methods for the synthesis, design, assembly, post synthesis processing, and application of synthetic DNA to modern biotechnology. Chapters are divided into three general sections focusing on protocols for the computational design of synthetic DNA sequences, the synthesis, assembly and cloning of synthetic DNA, and post-synthesis error reduction strategies. 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 tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Synthetic DNA: Methods and Protocols aims to help researchers further their research on manipulate DNA sequences.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Computational Tools for Design and Assembly of Synthetic DNA
Chapter 1: A Guide to Using STITCHER for Overlapping Assembly PCR Applications
1 Introduction
2 Materials
3 Methods
3.1 STITCHER Parameters
3.1.1 Input
3.1.2 Search Area
3.1.3 Primer GC% and Tm Calculation
3.1.4 Increments Along the Search Area
3.1.5 Self-­Complementarity and Repetitive Sequence
3.1.6 Overlap Fragment
3.1.7 Deletion Overlap
3.2 Remotely Retrieving Sequences
3.3 Off-Target Detection
3.4 Remote BLAST Searches
3.5 Checking Cross Complementarity
3.6 Literature Search on Overlapping PCR Applications
4 Notes
References
Chapter 2: Synthetic Gene Design Using Codon Optimization On-Line (COOL)
1 Introduction
2 Materials
2.1 Gene Design Criteria
2.2 Algorithm
2.3 Implementation
3 Methods
3.1 Input Sequence
3.2 Optimization Settings
3.3 Select Genes
3.4 Motif Settings
3.5 Submit
3.6 Check Results
3.7 Results
4 Case Study: Synthetic Gene Design of a Thermostable Invertase in E. coli
4.1 Input Sequence
4.2 Optimization Settings
4.3 Select Genes
4.4 Motif Settings
4.5 Submit
4.6 Results
5 Notes
References
Chapter 3: Shuffle Optimizer: A Program to Optimize DNA Shuffling for Protein Engineering
1 Introduction
2 Materials
2.1 For Running Shuffle Optimizer
2.2 For Shearing
2.3 For DNA Shuffling and General Reagents
3 Methods
3.1 Starting Sequence Considerations
3.2 Obtaining Sequences for Shuffle Optimizer Input
3.3 Running Shuffle Optimizer
3.4 Designing Primers and Constructs for DNA Shuffling
3.5 DNA Shuffling
4 Notes
References
Part II: DNA Synthesis, Assembly, and Cloning
Chapter 4: Simple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli
1 Introduction
2 Materials
2.1 Biological and Chemical Materials
2.2 Equipment
3 Methods
3.1 Primer Design
3.2 Simple Cloning
3.3 Easy Preparation of a Large-Size Random Gene Mutagenesis Library
4 Notes
References
Chapter 5: SpeedyGenes: Exploiting an Improved Gene Synthesis Method for the Efficient Production of Synthetic Protein Libraries for Directed Evolution
1 Introduction
2 Materials
3 Methods
3.1 Design of DNA Oligonucleotides
3.2 Assembly of Non-variant Sequences
3.2.1 Intermediate Block Synthesis
3.2.2 Endonuclease Digestion for Error Correction
3.2.3 Assembly of the Full-Length Sequence
3.3 Assembly of Variant Libraries
3.4 Downstream Cloning and E. coli Transformation
3.5 Concluding Remarks
4 Notes
References
Chapter 6: BASIC: A Simple and Accurate Modular DNA Assembly Method
1 Introduction
2 Materials
3 Methods
3.1 BASIC Part Preparation
3.2 Linker Preparation
3.3 Linker Ligation Reaction
3.4 Part Purification Step
3.5 DNA Assembly Step
3.6 Transformation of Assembled DNA
3.7 Screening for Correct Assemblies
4 Notes
References
Chapter 7: Enzymatic Synthesis of Single-Stranded Clonal Pure Oligonucleotides
1 Introduction
2 Materials
2.1 Equipment
2.2 RCA Strategy
2.3 Helper Phage Strategy
3 Methods
3.1 In Silico Pseudogene Design and E. coli Colony Preparation
3.2 RCA Strategy
3.3 Helper Phage Strategy
4 Notes
References
Chapter 8: Rapid Assembly of DNA via Ligase Cycling Reaction (LCR)
1 Introduction
2 Materials
2.1 Reagents for DNA Part Amplification, Purification, and Assembly
2.2 Equipment
3 Methods
3.1 Design of Bridging Oligos
3.2 Preparation of DNA Parts for Assembly
3.3 DNA Assembly via Ligase Cycling Reaction
3.4 Transformation to E. coli, Colony Counting, and Restriction Endonuclease DNA Fragment Analysis
4 Notes
References
Chapter 9: PaperClip: A Simple Method for Flexible Multi-Part DNA Assembly
1 Introduction
2 Materials
2.1 Clip Preparation
2.2 Polyacrylamide Gel Electrophoresis (for Optional Testing of Ligation Efficiency)
2.3 PCR and PCR Cleanup (for Optional Amplification of Parts Prior to Assembly)
2.4 Agarose Gel Electrophoresis (for Testing of Optional PCR Amplification)
2.5 Cell Extract Preparation
2.6 Assembly by PCR (Not Required if Assembly Is to Be Performed with Cell Extracts)
2.7 Bacterial Transformation
3 Methods
3.1 General Considerations in Planning the Assembly
3.2 Design of Clip Oligonucleotides
3.3 Phosphorylation and Annealing to Generate Half-Clips
3.4a Clip Ligation Without Insertion of Extra Elements Between Parts
3.5 3.4b Clip Ligation with Insertion of Extra Elements Between Parts
3.6 PAGE Analysis of Clip Ligation Efficiency (Optional but Recommended)
3.7 DNA Part Preparation
3.8 Agarose Gel Separation of Amplified Parts
3.9 Purification of the DNA Parts
3.10 Assembly by PCR
3.11 Preparation of Cell Extracts
3.12 Cell Extract-
3.13 Transformation
3.14 Selection of the Correct Clones
4 Notes
References
Chapter 10: The Polymerase Step Reaction (PSR) Method for Gene and Library Synthesis
1 Introduction
2 Materials
2.1 Primer Design
2.2 Primer Synthesis and Purification
2.3 Reagents and Equipment
3 Methods
3.1 PSR Assembly Reaction
3.2 Complementary Strand Fill-In
4 Notes
References
Chapter 11: Clonetegration Using OSIP Plasmids: One-Step DNA Assembly and Site-Specific Genomic Integration in Bacteria
1 Introduction
2 Materials
3 Methods
3.1 Generating and Assembling DNA Fragments
3.2 Transformation of the Assembly Product
3.3 Screening for Correct Integration
3.4 Removing the OSIP Backbone Using pE-FLP
4 Notes
References
Chapter 12: Generation of DNA Constructs Using the Golden GATEway Cloning Method
1 Introduction
2 Materials
2.1 Enzymes
2.2 Chemicals
2.3 Buffers and Media
2.4 Equipment and Kits
3 Methods
3.1 Cloning into Entry Vectors
3.1.1 Cloning into Entry Vectors via BamHI and KpnI
3.1.2 Cloning into Entry Vectors via TA Cloning
3.1.3 Cloning Oligonucleotides into Entry Vectors
Cloning of a Single-Oligo Duplex
Cloning of Multiple-Oligo Sets
3.1.4 Site-Directed Mutagenesis Using Golden Gate Cloning
3.2 Golden Gate Reaction
3.3 Further Use of the Assembled Construct
3.3.1 Multisite Gateway Cloning
3.3.2 Golden Gate Cloning Assembly via XcmI Back to pEVs
4 Notes
References
Chapter 13: Gene Deletion by Synthesis in Yeast
1 Introduction
2 Materials
2.1 Deletion Cassette and Oligonucleotide Design
2.2 Ligase Chain Reaction
2.3 Nested PCR
2.4 Yeast Transformation
2.5 Check PCR
2.6 Tetrad Analysis
3 Methods
3.1 Deletion Cassette and  Oligonucleotide Design
3.2 Ligase Chain Reaction
3.3 Nested PCR
3.4 Yeast Transformation (According to [15])
3.5 Check PCR
3.6 Tetrad Analysis (According to [15])
4 Notes
References
Chapter 14: Efficient Assembly of DNA Using Yeast Homologous Recombination (YHR)
1 Introduction
2 Materials
2.1 Reagents for DNA Part Amplification, Purification, and Assembly
2.2 Equipment
3 Methods
3.1 Design of Bridging Oligonucleotides
3.2 Preparation of DNA Parts for Assembly
3.3 DNA Assembly via Yeast Homologous Recombination
3.4 Transformation to E. coli, Colony Counting, and Restriction Endonuclease DNA Fragment Analysis
4 Notes
References
Chapter 15: Simultaneous Removal of Multiple DNA Segments by Polymerase Chain Reactions
1 Introduction
2 Materials
2.1 Components for Two-Step PCR Reaction
2.2 Components for Confirmation of Successful Removal of DNA Segments
3 Methods
3.1 Primer Design
3.2 Linearization by the First-­Step PCR
3.3 Clean-Up of the PCR Product
3.4 Recircularization by the Second-­Step PCR
3.5 Transformation and DNA Amplification
3.6 Confirmation of Successful Removal of DNA Segments by Blue/White Colony Screen Assay
3.7 Confirmation of Successful Removal of DNA Segments by Colony PCR Reactions
4 Notes
References
Chapter 16: Rapid Construction of Recombinant Plasmids by QuickStep-Cloning
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Nucleic Acid
2.3 PCR Components
2.4 DpnI Digestion and DNA Purification
2.5 Transformation and Clone Analysis
3 Methods
3.1 Primer Design
3.2 Asymmetric PCR
3.3 Megaprimer PCR
3.4 Transformation
3.5 Colony Analysis
4 Notes
References
Part III: Reducing Error in Synthetic DNA
Chapter 17: Immobilized MutS-Mediated Error Removal of Microchip-­Synthesized DNA
1 Introduction
2 Materials
2.1 Mcp-Oligo Subpool Preparation (See Note 1)
2.2 DNA Re-annealing
2.3 MutS Fusion Protein Preparation
2.4 Regenerated Amorphous Cellulose (RAC) Slurry Preparation
2.5 Error-Depleted Oligo Subpool Preparation
2.6 Error-Depleted Oligo Subpool Primer Removal
2.7 Oligo Subpool Assembly Components
3 Methods
3.1 Preparation of Mcp-Oligo Subpool
3.1.1 Design and Synthesis of the Mcp-­Oligo Pool
3.1.2 Mcp-Oligo Subpool Amplification
3.1.3 Re-annealing The Subpool PCR Products
3.2 Preparation of MutS Fusion Protein
3.2.1 Expression of the MutS Fusion Protein
3.2.2 Purification of the MutS Fusion Protein
3.2.3 Dialysis of the MutS Fusion Protein
3.2.4 Functional Evaluation of the MutS Fusion Proteins
3.3 Preparation of the RAC Slurry
3.4 Preparation of MICC
3.4.1 Pretreatment of RAC Slurry
3.4.2 Packing of MICC
4 Error Correction by MICC
4.1 Preparation of the Error-­Depleted Oligo Subpool
4.1.1 PCR Amplification of the Error-­Depleted Oligo Subpool
4.1.2 Primer Removal from the Error-­Depleted Oligo Subpool
4.2 Assembly of the Oligo Subpool
4.2.1 PCA Reaction
4.2.2 LCR Reaction
4.2.3 LCR-PCA Method
4.2.4 Amplification of the Assembled DNA Segments
5 Notes
References
Chapter 18: Selection of Error-Less Synthetic Genes in Yeast
1 Introduction
2 Materials
2.1 Gene Synthesis
2.2 Preparation of Linearized Vector DNA
2.3 Selection of Frame-Shift-
3 Methods
3.1 Gene Synthesis
3.2 Preparation of Linearized Vector DNA
3.3 Selection of Frame-Shift-
4 Notes
References
Index