Contents
- Preface
- Acknowledgments
- Contributors
- Abbreviations and Acronyms
1. Purifying and Analyzing Genomic DNA
- OVERVIEW OF GENOMIC DNA ANALYSIS
- Historical Perspective
- Issues of Contamination and Safety
- ISOLATION OF GENOMIC DNA
- DNA FROM MAMMALIAN SOURCES
- PROTOCOLS
- DNA Isolation from Tissue-culture Cell Lines Growing in Suspension
- Transforming Lymphoblasts with EBV
- Maintaining B95-8 and IA-3 EBV-producing Cell Lines
- DNA Isolation from Adherent Fibroblasts Using Trypsin
- DNA Isolation from Adherent Fibroblasts ("Sliming")
- DNA Isolation from Peripheral Blood
- DNA Isolation from Fresh and Frozen Tissues
- DNA Isolation from a Mammalian Model Organism: The Mouse
DNA FROM ANIMAL MODEL ORGANISMS
- PROTOCOLS
DNA Isolation from D. melanogaster
DNA Isolation from C. elegans
DNA FROM PLANTS
- PROTOCOL
DNA Isolation from Arabidopsis thaliana
DNA FROM YEAST
- PROTOCOLS
DNA Isolation from S. cerevisiae
DNA Isolation from S. pombe
DNA FROM BACTERIA
- PROTOCOL
DNA Isolation from E. coli
TROUBLESHOOTING FOR GENOMIC DNA ISOLATION
- Degradation and Fragmentation of the DNA
- Poor Recovery of DNA
- Contamination of the DNA with Proteins
- Contamination of the DNA with RNA
SOUTHERN BLOTTING
DIGESTION OF DNA WITH RESTRICTION ENZYMES
- PROTOCOL
Digesting Genomic DNA with Restriction Enzymes
ELECTROPHORETIC SEPARATION OF DIGESTION PRODUCTS
- PROTOCOL
Size Fractionating Digested DNA on Agarose Gels
TRANSFER OF DNA ONTO SOLID SUPPORTS
- PROTOCOL
Transferring Size-fractionated DNA onto Solid Supports
HYBRIDIZATION OF PROBES TO IMMOBILIZED DNA
- PROTOCOL
Gel Purification of DNA Fragments for Use as Probes
RADIOLABELING DNA PROBES
- PROTOCOLS
Radiolabeling Probes by Random Priming with Hexameric Oligonucleotides
End-labeling Oligonucleotide Probes
BLOCKING REPETITIVE DNA SEQUENCES
- PROTOCOLS
Preannealing Probes to Cot 1 Blocking DNA
Preannealing Probes to Genomic Blocking DNA
DETECTING UNIQUE SEQUENCES ON MEMBRANES BY USING RADIOLABELED PROBES
- PROTOCOLS
Hybridization Using Probes Labeled by Random Priming
Hybridization Using End-labeled Oligonucleotide Probes
USE OF NONRADIOACTIVE PROBES FOR HYBRIDIZATION
- Biotinylation of Probe DNA
- Membrane Preparation
- Hybridization and Washing
- Detection of Chemiluminescence
TROUBLESHOOTING FOR SOUTHERN BLOTTING
- Problems with the DNA Sample
- Problems with the Gel
- Problems with Transferring the DNA onto the Solid Support
- Problems with Hybridizing Radioactive or Nonradioactive Probes to the DNA
2. Preparation, Manipulation, and Mapping of HMW DNA
PRINCIPLES OF PREPARING AND MANIPULATING HMW DNA
- General Characteristics of HMW DNA
- Principles in the Preparation of HMW DNA
- Assessing the Quality of HMW DNA
- Quantitating HMW DNA
PREPARATION OF HMW MAMMALIAN DNA
- PROTOCOLS
Preparing Mononuclear Cells from Whole Blood by Centrifugation on Histopaque
Isolating White Blood Cells from Whole Blood by Sedimentation After Lysis of Red Blood Cells
Preparing HMW DNA in Agarose Starting from Frozen Whole Blood
Preparing Cells from Tissues
Preparing Cells from Tissue Culture
Preparing Cells from Frozen Semen
Using Proteinase K to Prepare HMW Mammalian DNA Embedded in Agarose Plugs
Using LIDS to Prepare HMW Mammalian DNA Embedded in Agarose Plugs
Preparing HMW Mammalian DNA in Agarose Beads
Purifying HMW Mammalian DNA in Liquid Form
PREPARATION OF HMW YEAST DNA
- PROTOCOLS
Growing S. cerevisiae Cells
Using Proteinase K to Prepare HMW Yeast DNA Embedded in Agarose Plugs
Using LIDS to Prepare HMW Yeast DNA Embedded in Agarose Plugs
Preparing HMW Yeast DNA in Agarose Beads
Purifying HMW Yeast DNA in Liquid Form
PREPARATION of HMW DNA FROM BACTERIAL CLONES
- PROTOCOLS
Preparing HMW Bacterial DNA Embedded in Agarose Plugs
Preparing HMW Bacterial DNA in Agarose Beads
DIGESTION OF HMW DNA WITH RESTRICTION ENZYMES
COMPLETE DIGESTION OF HMW DNA SAMPLES
- PROTOCOLS
Complete Digestion of HMW DNA Embedded in Agarose Plugs
Complete Digestion of HMW DNA Embedded in Agarose Beads
Complete Digestion of HMW DNA in Liquid Form
PARTIAL DIGESTION OF HMW DNA SAMPLES
- PROTOCOLS
Partial Digestion by Varying the Concentration of Restriction Enzyme
Partial Digestion by Varying the Length of the Incubation with the Restriction Enzyme
Partial Digestion by Varying the Mg++ Concentration
Partial Digestion by Methylase Competition
ANALYSIS AND PREPARATIVE SIZE FRACTIONATION OF PARTIAL DIGESTION PRODUCTS
- PROTOCOLS
Size Fractionation of Partial Digestion Fragments Using a Sucrose Density Gradient
Concentration of Liquid HMW DNA Fragments by Vacuum Dialysis
LONG-RANGE RESTRICTION MAPPING
- General Principles for Long-range Restriction Mapping
- Selection of Enzymes for Generating Large DNA Fragments
- Mapping Reagents
- Construction of a Long-range Restriction Map
- Troubleshooting for Long-range Restriction Mapping
SITE-SPECIFIC CLEAVAGE OF DNA USING RARE CLEAVAGE
- General Principles of RARE Cleavage
- Applications of RARE Cleavage
PROTOCOLS FOR RARE CLEAVAGE
- PROTOCOLS
RARE Cleavage with EcoRI or AluI in Low-percentage-agarose Plugs
RARE Cleavage with EcoRI in Diced Agarose Plugs
RARE Cleavage with EcoRI in Agarose Beads
- Potential Problems and Troubleshooting for RARE Cleavage
PULSED-FIELD GEL ELECTROPHORESIS
- Conventional Agarose Gel Electrophoresis and the Need for PFGE
- Setting Up the Components of a PFGE System
- Achieving Optimal Separation of DNAs
- Selecting Appropriate Electrophoresis Conditions
- Estimating DNA Fragment Sizes on a PFG
- Potential Problems with PFGE and Troubleshooting
SETTING UP AND RUNNING A PFG
- PROTOCOLS
Preparing the Gel and Loading DNA Samples in Agarose Beads or Plugs
Casting the Gel Around DNA Samples in Agarose Plugs
Preparing the Gel and Loading Liquid DNA Samples or Samples in Agarose Beads in a Submerged Gel
Running and Staining the Gel
PREPARATIVE PFGE FOR RECOVERY OF HMW DNA
RECOVERING HMW DNA FROM A PREPARATIVE PFG
- PROTOCOLS
Recovery of DNA from PFGs for Applications That Do Not Require Intact HMW DNA
Recovery of Intact HMW DNA from PFGs
- Troubleshooting for the Recovery of Intact HMW DNA
- Handling and Treatment of Gel-purified HMW DNA for Transfer into Mammalian Cells
3. PCR in Genome Analysis
BASIC PRINCIPLES AND COMPONENTS OF PCR
- A Simple PCR Protocol
- Oligonucleotide Primers
- Sample Preparation
- Thermostable DNA Polymerases
- Instrumentation
BASIC PROTOCOLS NEEDED FOR PREPARING KEY COMPONENTS OF THE PCR ASSAY
- PROTOCOLS
- Basic DNA Amplification
- Desalting Oligonucleotides by Precipitation
- Desalting Oligonucleotides on Nensorb Affinity Columns
- Rapid Preparation of Template DNA from Mammalian Cells
OPTIMIZING PCR ASSAYS
- Selecting New Primers
- Modifying Thermal Cycling Conditions
- Altering the Mg++ Concentration
- Altering the pH
- Altering Other Reaction Components
- Troubleshooting
METHODS FOR IMPROVING PCR
- Hot Start PCR
- Nested PCR
- Touch-down PCR
- Booster PCR
DETECTING AND CHARACTERIZING PCR PRODUCTS
- Detection Schemes
- Quantitative PCR
- Cloning PCR Products
- Amplification Using Mixed Oligonucleotides as Primers
- In Vitro Gene Assembly by PCR
- RT-PCR for Amplification from RNA
- In Situ PCR
- Amplification of Large DNA Segments (Long-range PCR)
- PCR-based Fingerprinting
- Multiplex PCR
PROTOCOLS FOR DETECTING AND CHARACTERIZING PCR PRODUCTS
- PROTOCOLS
- Long-range PCR Protocol
- DD PCR Protocol
CONTAMINATION IN PCR ASSAYS
- Sources of Contamination
- Avoidance of Contamination
- Elimination of Contamination
FURTHER READING
4. Fluorescence-based DNA Sequencing
OVERVIEW OF DNA SEQUENCING METHODS
PREPARING SUBCLONED DNA TEMPLATES
- PROTOCOLS
- Preparing Single-stranded Bacteriophage M13 DNA Using PEG and Phenol
- Preparing Single-stranded Bacteriophage M13 DNA Using Detergent
- Preparing Single-stranded Phagemid DNA
- Purifying Double-stranded Plasmid DNA by Alkaline Lysis
- Purifying Double-stranded Plasmid DNA in a 96-well Format Using an Alkaline Lysis Kit
- Denaturing Double-stranded DNA for Sequencing
- Denaturing Double-stranded DNA and Annealing Primer to Template for Sequencing
PREPARING SEQUENCING TEMPLATES BY PCR
- PROTOCOLS
- Amplifying Subclone Inserts by PCR and Precipitating PCR Products with PEG to Purify Sequencing Template
- Treating PCR Products with Exonuclease I plus Shrimp Alkaline Phosphatase to Purify Sequencing Template
- Preparing Single-stranded Template DNA by Asymmetric PCR
- Preparing Single-stranded Template DNA from PCR Products Using Bacteriophage T7 Gene 6 Exonuclease
- Preparing Single-stranded Template DNA from PCR Products Using Bacteriophage lambda Exonuclease
- Purifying Single-stranded Template DNA from PCR Products Using Streptavidin-linked Magnetic Beads
SEQUENCING PRIMERS
FLUORESCENCE-BASED DNA SEQUENCING REACTIONS
- PROTOCOLS
- Sequencing Reactions Using Fluorescent Dye-labeled Primers and Sequenase DNA Polymerase
- Sequencing Reactions Using Fluorescent Dye-labeled Primers and ThermoSequenase DNA Polymerase
- Sequencing Reactions Using Fluorescent Dye-labeled Primers and SequiTherm DNA Polymerase
- Sequencing Reactions Using Fluorescent Dye-labeled Terminators and ThermoSequenase DNA Polymerase
- Sequencing Reactions Using Fluorescent Dye-labeled Terminators and AmpliTaq DNA Polymerase FS
- Removing Unincorporated Dye-labeled Terminators after Cycle Sequencing by Using Sephadex in Filter Plates
- Removing Unincorporated Dye-labeled Terminators after Cycle Sequencing by Using Centri-Sep Columns
PREPARING, LOADING, AND RUNNING GELS ON FLUORESCENCE-BASED SEQUENCING INSTRUMENTS
- PROTOCOLS
- Washing and Assembling Gel Plates
- Pouring the Gel
- Loading and Running the Gel
TROUBLESHOOTING FOR FLUORESCENCE-BASED DNA SEQUENCING
- Problems Caused by Software
- Problems Caused by Templates or by Pipetting Errors
- Problems Caused by DNA Structure
STRATEGIES FOR DETERMINING CONTIGUOUS SEGMENTS OF DNA SEQUENCE
- Types of Strategies
- Choosing a Strategy
PRIMER-DIRECTED SEQUENCING
DELETIONAL SEQUENCING
- PROTOCOLS
- Digestion of Template DNA with Restriction Enzymes
- Digestion of Template DNA with Exonuclease III and Mung-bean Nuclease
- Religation of Subclones That Have Deletions and Transformation of
E. coli
SEQUENCING cDNA CLONES
5. Shotgun Sequencing
OVERVIEW OF SHOTGUN SEQUENCING
LIBRARY CONSTRUCTION
PREPARATION OF TARGET DNA
- PROTOCOLS
- Purification of Bacterial Clone DNA on CsCl Gradients
- Preparation of Bacterial Clone DNA by Gentle Alkaline Lysis
- Purification of Agarose-embedded HMW Yeast DNA for Isolation of YAC DNA by Preparative PFGE
FRAGMENTATION OF TARGET DNA
- PROTOCOLS
- Random Fragmentation by Low-pressure Shearing in a French Pressure Cell
- Random Fragmentation by Sonication
- Random Fragmentation by Nebulization
- Random Fragmentation with DNase I
- Random Fragmentation by Partial Digestion with Sau3AI
END REPAIR AND SIZE FRACTIONATION OF FRAGMENTED DNA
- PROTOCOLS
- Using DNA Polymerases and Polynucleotide Kinase to Repair Fragment Ends
- Using Mung-bean Nuclease to Repair Fragment Ends
- Size Fractionation of Repaired DNA Fragments
CLONING FRAGMENTED DNA
- PROTOCOLS
- Vector Preparation
- Ligation of Vector DNA and Insert DNA
- Transforming Bacteria with Ligated DNA by Using Electroporation
- Preparation of Cells Competent for Electroporation
- Electroporation
- Transforming Bacteria with Ligated DNA by Using CaCl2 and Heat Shock
- Preparation of Competent Cells
- Transformation
INITIAL SEQUENCE ACQUISITION, DATA PROCESSING, AND SEQUENCE ASSEMBLY
SEQUENCE FINISHING
- Subclone-directed Closure
- Primer-directed Closure
- Resolving Ambiguities
- Resolving Repeats
6. Transposon-mediated DNA Sequencing
COMPARISON OF SHOTGUN SEQUENCING AND TRANSPOSON-MEDIATED SEQUENCING
TRANSPOSONS USED FOR DNA SEQUENCING
gamma delta TRANSPOSON-MEDIATED DNA SEQUENCING
PREPARING LIBRARIES OF SUBCLONES WITH 3 - 4-KB INSERTS FROM LARGE GENOMIC CLONES
- PROTOCOLS
- Sonication, End Repair, and Size Selection of 3 - 4-kb Subclone Inserts
- Preparation of BstXI-digested pOT2A Vector DNA
- Addition of BstXI Adapters to Size-selected Insert DNA and Ligation to BstXI-digested Vector DNA
BUILDING INITIAL SUBCLONE TILING PATHS
- PROTOCOL
- Building Initial Subclone Tiling Paths from End Sequences
SEQUENCING SUBCLONES BY USING THE gamma delta TRANSPOSON
- PROTOCOLS
- Mobilizing Transposons into Subclones
- Mapping gamma delta Transposon-insertion Sites by Using PCR
- Sequencing the Minimal Set of Transposon-bearing Templates
- Assembling Subclone Sequences
EXTENDING SUBCLONE TILING PATHS AND ASSEMBLING SUBCLONE SEQUENCES INTO LARGER CONTIGS
- PROTOCOL
- Extending Stalled Contigs in the Tiling Path and Assembling the Finished Genomic Clone Sequence
IN VITRO TY1 TRANSPOSON-MEDIATED DNA SEQUENCING
INTEGRATION OF ARTIFICIAL TRANSPOSONS INTO TARGET DNA IN VITRO
- PROTOCOLS
- Preparation of AT-2 for In Vitro Integration
- Purification of VLPs for In Vitro Integration
- Growing Gal-Ty1 Yeast Strains and Inducing the GAL1 Promoter
- Recovering and Lysing Cells
- Centrifuging the Lysate on a Sucrose Gradient and Identifying Fractions with Integrase Activity
- In Vitro Integration of AT-2 into Target DNA
- Recovery of Recombinant Plasmids by Electroporation and Selection with Antibiotics
SEQUENCING FROM MAPPED OR RANDOMLY CHOSEN TRANSPOSON-INSERTION SITES
- PROTOCOLS
- Mapping Ty1 Transposon-insertion Sites by Using PCR
- Restriction Mapping of Ty1 Transposon-insertion Sites
- Sequencing DNA from Sites of AT-2 Insertion
TROUBLESHOOTING FOR IN VITRO TY1 TRANSPOSON-MEDIATED SEQUENCING
- Evaluation of Integration Efficiency
- Optimization of Molar Ratios of Transposon DNA to Target DNA
- Cotransformation with Parental Target Plasmids and Recombinants
- Contamination by Recombinants from Previous Experiments
7. Computational Analysis of DNA and Protein Sequences
[Selected Chapter online]
INTERNET BASICS
- Electronic Mail
- File Transfer Protocol
- Gopher
- The World Wide Web
- Electronic Publishing
- Specialized Client-Server Applications
SEQUENCE ANALYSIS
- Searching Databases for Similarities
- Sequence Databases
- The BLAST Family of Programs: Uses and Examples
- DNA Query Sequences
- Protein Query Sequences
- Confounding Subsequences and Query Masking
- Additional BLAST Options
INTEGRATED INFORMATION RETRIEVAL
- The Entrez System
- Specialized Data Sets
- C. elegans
- D. melanogaster
MULTIPLE ALIGNMENT, SEQUENCE MOTIFS, AND STRUCTURE INFERENCE
- Multiple Alignment
- Sequence Motifs
- Structure Prediction and Protein Modeling
SUBMITTING DATA TO PUBLIC DATABASES
- Preparing a New Submission
- Updates and Corrections
- Special Arrangements for Large Projects
APPENDICES
1. Common Reagents
Standard Stock Solutions
Molarities of Concentrated Acids and Bases
Common Laboratory Solutions
Electrophoresis Buffers, Dyes, and Gel-loading Solutions
Media
Antimicrobial Agents
2. Basic Procedures
Quantitation of Cell Concentration
Standard Methods Used for Isolating DNA
- Extraction of DNA Samples with Organic Chemicals
- Concentration of DNA Samples by Precipitation with Alcohols
- Quantitation of DNA
- Dialysis of DNA
Assessing the Extent of Radiolabeling in DNA Probes by Precipitation with TCA
Dilution and Storage of Oligonucleotides
Storage and Shipment of Biological Samples
3. Safety Cautions
4. Useful Facts
Conversion Factors
Genome Comparisons
Average Sizes of DNA Fragments Generated by Cleavage with Restriction Enzymes
Single-letter Abbreviations for Amino Acids
Genetic Code
Codon Facts
Isotope Information
5. Suppliers
Index
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