Current Protocols in Molecular Biology

Foreword by Phillip A. Sharp
Preface
Contributors

1 Escherichia coli, Plasmids, and Bacteriophages
I ESCHERICHIA COLI
1.1 Media Preparation and Bacteriological Tools
1.2 Growth in Liquid Media
1.3 Growth on Solid Media)
1.4 Classical Bacterial Genetics
II VECTORS DERIVED FROM PLASMIDS
1.5 Introduction to Plasmid Biology
1.6 Minipreps of Plasmid DNA
1.7 Large-Scale Preparation of Plasmid DNA
1.8 Introduction of Plasmid DNA into Cells
III VECTORS DERIVED FROM LAMBDA AND RELATED BACTERIOPHAGES
1.9 Introduction to Lambda Phages
1.10 Lambda as a Cloning Vector
1.11 Plating Lambda Phage to Generate Plaques
1.12 Growing Lambda-Derived Vectors
1.13 Preparing Lambda DNA from Phage Lysates
IV VECTORS DERIVED FROM FILAMENTOUS PHAGES
1.14 Introduction to Vectors Derived from Filamentous Phages
1.15 Preparing and Using M13-Derived Vectors
1.16 Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

2 Preparation and Analysis of DNA
I PREPARATION OF GENOMIC DNA
2.1A Purification and Concentration of DNA
2.1B Purification of DNA by Anion-Exchange Chromatography
2.2 Preparation of Genomic DNA from Mammalian Tissue
2.3 Preparation of Genomic DNA from Plant Tissue
2.4 Preparation of Genomic DNA from Bacteria
II RESOLUTION AND RECOVERY OF LARGE DNA FRAGMENTS
2.5A Agarose Gel Electrophoresis
2.5B Pulsed-Field Gel Electrophoresis
2.6 Purification of Large DNA Fragments
III RESOLUTION AND RECOVERY OF SMALL DNA FRAGMENTS
2.7 Separation of Small DNA Fragments by Conventional Gel Electrophoresis
2.8 Capillary Electrophoresis of DNA
IV ANALYSIS OF DNA SEQUENCES BY BLOTTING AND HYBRIDIZATION
2.9A Southern Blotting
2.9B Dot and Slot Blotting of DNA
2.10 Hybridization Analysis of DNA Blots
V SYNTHESIS AND PURIFICATION OF OLIGONUCLEOTIDES
2.11 Synthesis of Oligonucleotides
2.12 Purification of Oligonucleotides Using Denaturing Polyacrylamide Gel Electrophoresis

3 Enzymatic Manipulation of DNA and RNA
I RESTRICTION ENDONUCLEASES
3.1 Digestion of DNA with Restriction Endonucleases
II RESTRICTION MAPPING
3.2 Mapping by Multiple Endonuclease Digestions
3.3 Mapping by Partial Endonuclease Digestions
III ENZYMES FOR MODIFYING AND RADIOACTIVELY LABELING NUCLEIC ACIDS
3.4 Reagents and Radioisotopes Used to Manipulate Nucleic Acids
3.5 DNA-Dependent DNA Polymerases
3.6 Template-Independent DNA Polymerases
3.7 RNA-Dependent DNA Polymerases
3.8 DNA-Dependent RNA Polymerases
3.9 DNA-Independent RNA Polymerases
3.10 Phosphatases and Kinases
3.11 Exonucleases
3.12 Endonucleases
3.13 Ribonucleases
3.14 DNA Ligases
3.15 RNA Ligases
IV CONSTRUCTION OF HYBRID DNA MOLECULES
3.16 Subcloning of DNA Fragments
3.17 Constructing Recombinant DNA Molecules by the Polymerase Chain Reaction
V SPECIALIZED APPLICATIONS
3.18 Labeling and Colorimetric Detection of Nonisotopic Probes
3.19 Chemiluminescent Detection of Nonisotopic Probes

4 Preparation and Analysis of RNA
I PREPARATION OF RNA FROM EUKARYOTIC AND PROKARYOTIC CELLS
4.1 Preparation of Cytoplasmic RNA from Tissue Culture Cells
4.2 Guanidine Method for Total RNA Preparation
4.3 Phenol/SDS Method for Plant RNA Preparation
4.4 Preparation of Bacterial RNA
4.5 Preparation of Poly(A)+ RNA
II ANALYSIS OF RNA STRUCTURE AND SYNTHESIS
4.6 S1 Analysis of Messenger RNA Using Single-Stranded DNA Probes
4.7 Ribonuclease Protection Assay
4.8 Primer Extension
4.9 Analysis of RNA by Northern and Slot Blot Hybridization
4.10 Identification of Newly Transcribed RNA

5 Construction of Recombinant DNA Libraries
I OVERVIEW OF RECOMBINANT DNA LIBRARIES
5.1 Genomic DNA Libraries
5.2 cDNA Libraries
II PREPARATION OF INSERT DNA FROM GENOMIC DNA
5.3 Size Fractionation Using Sucrose Gradients
5.4 Size Fractionation Using Agarose Gels
III PREPARATION OF INSERT DNA FROM MESSENGER RNA
5.5 Conversion of mRNA into Double-Stranded cDNA
5.6 Methylation and Addition of Linkers to Double-Stranded cDNA
IV PRODUCTION OF GENOMIC DNA AND cDNA LIBRARIES
5.7 Production of a Genomic DNA Library
5.8A Production of a Complete cDNA Library
5.8B Production of a Subtracted cDNA Library
5.9 Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
V AMPLIFICATION OF TRANSFORMED OR PACKAGED LIBRARIES
5.10 Amplification of a Bacteriophage Library
5.11 Amplification of Cosmid and Plasmid Libraries

6 Screening of Recombinant DNA Libraries
I PLATING LIBRARIES AND TRANSFER TO FILTER MEMBRANES
6.1 Plating and Transferring Bacteriophage Libraries
6.2 Plating and Transferring Cosmid and Plasmid Libraries
II HYBRIDIZATION WITH RADIOACTIVE PROBES
6.3 Using DNA Fragments as Probes
6.4 Using Synthetic Oligonucleotides as Probes
III PURIFICATION OF BACTERIOPHAGE, COSMID, AND PLASMID CLONES
6.5 Purification of Bacteriophage Clones
6.6 Purification of Cosmid and Plasmid Clones
IV SCREENING WITH ANTIBODIES
6.7 Immunoscreening of Fusion Proteins Produced in Lambda Plaques
6.8 Immunoscreening after Hybrid Selection and Translation
6.9 Yeast Artificial Chromosome Libraries
6.10 Analysis of Isolated YAC Clones
VI SPECIALIZED STRATEGIES FOR SCREENING LIBRARIES
6.11 Use of Monoclonal Antibodies for Expression Cloning
6.12 Recombination-Based Assay (RBA) for Screening Bacteriophage Lambda Libraries

7 DNA Sequencing
7.1 DNA Sequencing Strategies
7.2 Construction of Nested Deletions for DNA Sequencing
7.3 Preparation of Templates for DNA Sequencing
7.4A DNA Sequencing by the Dideoxy Method
7.4B Dideoxy DNA Sequencing with Chemiluminescent Detection
7.5 DNA Sequencing by the Chemical Method
7.6 Denaturing Gel Electrophoresis for Sequencing
7.7 Computer Manipulation of DNA and Protein Sequences

8 Mutagenesis of Cloned DNA
8.1 Oligonucleotide-Directed Mutagenesis without Phenotypic Selection
8.2A Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations in a Small DNA Sequence
8.2B Gene Synthesis: Assembly of Target Sequences Using Mutually Priming Long Oligonucleotides
8.3 Random Mutagenesis by PCR
8.4 Linker-Scanning Mutagenesis of DNA
8.5 Directed Mutagenesis using the Polymerase Chain Reaction

9 Introduction of DNA into Mammalian Cells
I TRANSFECTION OF DNA INTO EUKARYOTIC CELLS
9.1 Calcium Phosphate Transfection
9.2 Transfection Using DEAE-Dextran
9.3 Transfection by Electroporation
9.4 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
9.5 Selection of Transfected Mammalian Cells
II USES OF FUSION GENES IN MAMMALIAN TRANSFECTION
9.6 Overview of Genetic Reporter Systems
9.7A Isotopic Assays for Reporter Gene Activity
9.7B Nonisotopic Assays for Reporter Gene Activity
9.7C Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics in Vivo
9.8 Direct Analysis of RNA After Transfection
III TRANSDUCTION OF GENES USING RETROVIRUS VECTORS
9.9 Overview of the Retrovirus Transduction System
9.10 Preparation of a Specific Retrovirus Producer Cell Line
9.11 Transient Transfection Methods for Preparation of High-Titer Retroviral Supernatants
9.12 Large-Scale Preparation and Concentration of Retrovirus Stocks
9.13 Detection of Helper Virus in Retrovirus Stocks
9.14 Retrovirus Infection of Cells In Vitro and In Vivo
IV GENE TARGETING BY HOMOLOGOUS RECOMBINATION
9.15 Human Somatic Cell Gene Targeting
9.16 Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)
9.17 Production of a Homozygous Mutant Embryonic Stem Cell Line (Double Knockout)

10 Analysis of Proteins
I QUANTITATION OF PROTEINS
10.1A Spectrophotometric and Colorimetric Determination of Protein Concentration
10.1B Quantitative Amino Acid Analysis
II ELECTROPHORETIC SEPARATION OF PROTEINS
10.2A One-Dimensional SDS Gel Electrophoresis of Proteins
10.2B One-Dimensional Gel Electrophoresis Using Nondenaturing Conditions
10.3 Two-Dimensional Gel Electrophoresis Using the ISO-DALT System
10.4 Two-Dimensional Gel Electrophoresis Using the O'Farrell System
10.5 Overview of Digital Electrophoresis Analysis
III DETECTION OF PROTEINS
10.6 Staining Proteins in Gels
10.7 Detection of Proteins on Blot Transfer Membranes (Revised, October 2003)
10.8 Immunoblotting and Immunodetection
IV PURIFICATION OF PROTEINS BY CONVENTIONAL CHROMATOGRAPHY
10.9 Gel-Filtration Chromatography
10.10 Ion-Exchange Chromatography
10.11A Immunoaffinity Chromatography
10.11B Metal-Chelate Affinity Chromatography
V PURIFICATION OF PROTEINS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
10.12 HPLC of Peptides and Proteins: Preparation and System Set-Up
10.13 HPLC of Peptides and Proteins: Standard Operating Conditions
10.14 Reversed-Phase Isolation of Peptides
10.15 Purification of Recombinant Proteins and Study of Protein Interaction by Epitope Tagging
VI PURIFICATION OF PROTEINS BY PRECIPITATION
10.16 Immunoprecipitation
VII SPECIALIZED APPLICATIONS
10.17 Synthesizing Proteins In Vitro by Transcription and Translation of Cloned Genes
10.18 Metabolic Labeling of Aminco Acids
10.19 Isolation of Proteins for Microsequence Analysis
10.20 Capillary Electrophoresis of Proteins and Peptides
10.21 Overview of Peptide and Protein Analysis by Mass Spectrometry
10.22 Protein Identification and Characterization by Mass Spectrometry

11 Immunology
I IMMUNOASSAYS
11.1 Conjugation of Enzymes to Antibodies
11.2 Enzyme-Linked Immunosorbent Assay (ELISA)
11.3 Isotype Determination of Antibodies
II PREPARATION OF MONOCLONAL ANTIBODIES
11.4 Immunization of Mice
11.5 Preparation of Myeloma Cells
11.6 Preparation of Mouse Feeder Cells for Fusion and Cloning
11.7 Fusion of Myeloma Cells with Immune Spleen Cells
11.8 Cloning of Hybridoma Cell Lines by Limiting Dilution
11.9 Freezing and Recovery of Hybridoma Cell Lines
11.10 Production of Monoclonal Antibody Supernatant and Ascites Fluids
11.11 Purification of Monoclonal Antibodies
III PREPARATION OF POLYCLONAL ANTISERA
11.12 Production of Polyclonal Antisera
11.13 In Vitro Antibody Production
11.14 Purification of Immunoglobulin G Fraction from Antiserum, Ascites Fluid, or Hybridoma Supernatants
IV PREPARATION OF ANTIPEPTIDE ANTIBODIES
11.15 Introduction to Peptide Synthesis
11.16 Synthetic Peptides for Production of Antibodies
V DETERMINATION OF SPECIFIC ANTIBODY TITER AND ISOTYPE
11.17 Determination of the Specific Antibody Titer
11.18 Identification of Polyol-Responsive MAbs for Use in Immunoaffinity Chromatography

12 DNA-Protein Interactions
12.1 Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
12.2 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis
12.3 Methylation and Uracil Interference Assays for Analysis of Protein-DNA Interactions
12.4 DNase I Footprint Analysis of Protein-DNA Binding
12.5 UV Crosslinking of Proteins to Nucleic Acids
12.6 Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems
12.7 Detection, Purification, and Characterization of cDNA Clones Encoding DNA-Binding Proteins
12.8 Rapid Separation of Protein-Bound DNA from Free DNA Using Nitrocellulose Filters
12.9 Analysis of DNA-Protein Interactions Using Proteins Synthesized In Vitro from Cloned Genes
12.10 Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography
12.11 Determination of Protein-DNA Sequence Specificity by PCR-Assisted Binding-Site Selection
12.12 Yeast One-Hybrid Screening for DNA-Protein Interactions

13 Saccharomyces cerevisiae
I BASIC TECHNIQUES OF YEAST GENETICS
13.1 Preparation of Yeast Media
13.2 Growth and Manipulation of Yeast
13.3 Genome-Wide Transposon Mutagenesis in Yeast
II YEAST VECTORS
13.4 Yeast Cloning Vectors and Genes
13.6 Yeast Vectors for Expression of Cloned Genes
III MANIPULATION OF YEAST GENES
13.7 Introduction of DNA into Yeast Cells
13.8 Cloning Yeast Genes by Complementation
13.9 Manipulation of Plasmids from Yeast Cells
13.10 Manipulation of Cloned Yeast DNA
IV PREPARATION OF YEAST DNA, RNA, AND PROTEINS
13.11 Preparation of Yeast DNA
13.12 Preparation of Yeast RNA
13.13 Preparation of Protein Extracts from Yeast
13.14 Overview of Schizosaccharomyces pombe (New, October 2003)
13.15 S. pombe Strain Maintenance and Media (New, October 2003)
13.16 Growth and Manipulation of S. pombe (New, October 2003)
13.17 Introduction of DNA into S. pombe Cells (New, October 2003)

14 In situ Hybridization and Immunohistochemistry

14.1 Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
14.2 Cryosectioning
14.3 In Situ Hybridization to Cellular RNA
14.4 Detection of Hybridized Probe
14.5 Counterstaining and Mounting of Autoradiographed In situ Hybridization Slides
14.6 Immunohistochemistry
14.7 In situ Hybridization and Detection Using Nonisotopic Probes
14.8 In situ Polymerase Chain Reaction and Hybridization to Detect Low-Abundance Nucleic Acid Targets
14.9 Whole-Mount in situ Hybridization and Detection of RNAs in Vertebrate Embryos and Isolated Organs
14.10 Principles and Applications of Fluorescence Microscopy
14.11 Basic Confocal Miscroscopy
14.12 Measurement of In Situ Hybridization
14.13 Morphological, Biochemical, and Flow Cytometric Assays of Apoptosis
14.14 Whole-Mount Histochemical Detection of b-Galactosidase Activity
14.15 Overview of Image Analysis, Image Importing, and Image Processing Using Freeware

15 The Polymerase Chain Reaction
15.1 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
15.2 Direct DNA Sequencing of PCR Products
15.3 Ligation-Mediated PCR for Genomic Sequencing and Footprinting
15.4 Molecular Cloning of PCR Products
15.5 Enzymatic Amplification of RNA by PCR (RT-PCR)
15.6 cDNA Amplification Using One-Sided (Anchored) PCR
15.7 Quantitation of Rare DNAs by PCR
15.8 Differential Display of mRNA by PCR

16 Protein Expression
I EXPRESSION OF PROTEINS IN ESCHERICHIA COLI
16.1 Overview of Protein Expression in E. coli
16.2 Expression Using the T7 RNA Polymerase/Promoter System
16.3 Expression Using Vectors with Phage l Regulatory Sequences
16.4A Introduction to Expression by Fusion Protein Vectors
16.4B Enzymatic and Chemical Cleavage of Fusion Proteins
16.5 Expression and Purification of lacZ and trpE Fusion Proteins
16.6 Expression and Purification of Maltose-Binding Protein Fusions
16.7 Expression and Purification of Glutathione-S-Transferase Fusion Proteins
16.8 Expression and Purification of Thioredoxin Fusion Proteins
II EXPRESSION OF PROTEINS IN INSECT CELLS USING BACULOVIRUS VECTORS
16.9 Overview of the Baculovirus Expression System (Revised, January 2004)
16.10 Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses (Revised, January 2004)
16.11 Expression and Purification of Recombinant Proteins Using the Baculovirus System ((Revised, January 2004)

III EXPRESSION OF PROTEINS IN MAMMALIAN CELLS
16.12 Transient Expression of Proteins Using COS Cells
16.13 Epitope Tagging and Expression
16.14 Inducible Gene Expression Using an Autoregulatory, Tetracyline-Controlled System
16.15 Overview of the Vaccinia Virus Expression System
16.16 Preparation of Cell Cultures and Vaccinia Virus Stocks
16.17 Generation of Recombinant Vaccinia Viruses
16.18 Characterization of Recombinant Vaccinia Viruses and Their Products
16.19 Gene Expression Using the Vaccinia/T7 RNA Polymerase Hybrid System
16.20 Expression of Proteins Using Semliki Forest Virus Vectors
16.21 Overview of the HIV-1 Lentiviral System
16.22 Generation of HIV-1-based Lentiviral Vector Particles
16.23 Amplification Using CHO Cell Expression Vectors

17 Preparation and Analysis of Glycoconjugates
I SPECIAL CONSIDERATIONS OF GLYCOCONJUGATES AND THEIR PURIFICATION
17.1 Special Considerations for Glycoproteins and Their Purification
17.2 Special Considerations for Proteoglycans and Glycosaminoglycans and Their Purification
17.3 Special Considerations for Glycolipids and Their Purification
II DETECTION OF SACCHARIDES ON GLYCOCONJUGATES
17.4 Metabolic Radiolabeling of Animal Cell Glycoconjugates
17.5 Chemical Labeling of Carbohydrates by Oxidation and Sodium Borohydride Reduction
17.6 Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine
17.7 Lectin Analysis of Proteins Blotted onto Filters
17.8 Detection of Glycophospholipid Anchors on Proteins
17.9 Direct Chemical Analysis of Glycoconjugates for Carbohydrates
17.10A Inhibition of N-Linked Glycosylation
17.10B Inhibition of Glycolipid Biosynthesis
17.11 Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and Inhibitors of Glycoprotein and Proteoglycan Assembly
III RELEASE OF SACCHARIDES FROM GLYCOCONJUGATES
17.12 Sialidases
17.13A Endoglycosidase and Glycoamidase Release of N-Linked Oligosaccharides
17.13B Analysis of Glycosaminoglycans with Polysaccharide Lyases
17.14A Preparation of Glycopeptides
17.14B Detection of Individual Glycosylation Sites on Glycoproteins
17.15A -Elimination for Release of O-Linked Glycosaminoglycans from Proteoglycans
17.15B -Elimination for Release of O-GalNAc-Linked Oligosaccharides from Glycoproteins and Glycopeptides
17.16 Acid Hydrolysis for Release of Monosaccharides
17.17A Enzymatic Release of Oligosaccharides from Glycolipids
17.17B Endo -Galactosidases and Keratanase
IV ANALYSIS OF SACCHARIDES RELEASED FROM GLYCOCONJUGATES
17.18 Analysis of Monosaccharides
17.19A Total Compositional Analysis by High-Performance Liquid Chromatography or Gas-Liquid Chromatography
17.19B Composition of Labeled Monosaccharides from Glycosaminoglycans
17.20 Analysis of Oligosaccharide Negative Charge by Anion-Exchange Chromatography
17.21A HPLC Methods for the Fractionation and Analysis of Negatively Charged Oligosaccharides and Gangliosides
17.21B Fractionation and Analysis of Neutral Oligosaccharides by HPLC
17.22A Nitrous Acid Degradation of Glycosaminoglycans
17.22B Analysis of Disaccharides and Tetrasaccharides Released from Glycosaminoglycans
17.23 Analysis of Sulfate Esters by Solvolysis or Hydrolysis

18 Analysis of Protein Phosphorylation
18.1 Overview of Protein Phosphorylation
18.2 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
18.3 Phosphoamino Acid Analysis
18.4 Analysis of Phosphorylation of Unlabeled Proteins
18.5 Detection of Phosphorylation by Enzymatic Techniques
18.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
18.7 Assays of Protein Kinases Using Exogenous Substrates
18.8 Permeabilization Strategies to Study Protein Phosphorylation
18.9 Phosphopeptide Mapping and Identification of Phosphorylation Sites
18.10 Use of Protein Phosphatase Inhibitors

19 Informatics for Molecular Biologists
19.1 Internet Basics for Biologists
19.2 Sequence Databases: Information Retrieval and Data Submission
19.3 Sequence Similarity Searching Using the BLAST Family of Programs
19.4 Protein Databases on the Internet

20 Analysis of Protein Interactions
20.1 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
20.2 Affinity Purification of Proteins Binding to GST Fusion Proteins
20.3 Phage-Based Expression Cloning
20.4 Surface Plasmon Resonance for Measurements of Biological Interest
20.5 Dectecton of Protein-Protein Interactions by Coprecipitation
20.6 Identification of Protein Interactions by Far Western Analysis
20.7 Two-Hybrid Dual Bait System
20.8 Interaction Trap/Two-Hybrid System to Identify Loss-of-Interaction Mutant Proteins (New, January 2004)

21 Chromatin Assembly and Analysis
21.1 Micrococcal Nuclease Analysis of Chromatin Structure
21.2 Separation of Histone Variants by TAU Gels and Isoforms
21.3 Chromatin Immunoprecipitation (Revised, January 2004)
21.4 DNase I and Hydroxyl Radical Characterization of Chromatin Complexes
21.5 Isolation of Histones and Nucleosome Cores from Mammalian Cells
21.6 Assembly of Nucleosomal Templates by Salt Dialysis
21.7 Chromatin Assembly Using Drosophila Systems

22 Nucleic Acid Arrays
22.1 Overview of Nucleic Acid Arrays
22.2 Preparation of mRNA for Expression Monitoring
22.3 Profiling Human Gene Expression with cDNA Microarrays

23 Manipulating the Mouse Genome

23.1 Overview of Gene Targeting by Homologous Recombination
23.2 Mouse Embryo Fibroblast (MEF) Feeder Cell Preparation
23.3 Mouse Embryonic Stem (ES) Cell Culture
23.4 Mouse Embryonic Stem (ES) Cell Isolation
23.5 Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)
23.6 Production of a Homozygous Mutant Embryonic Stem Cell Line (Double Knockout)
23.7 Chimeric Mouse Production by Microinjection
23.8 Mouse Colony Management

24 Generation and Use of Combinatorial Libraries

24.1 Coming soon!
24.2 Design, Synthesis, and Amplification of DNA Pools for Construction of Combinatorial Pools and Libraries
24.3 In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization
24.4 Peptide Aptamers: Dominant "Genetic" Agents for Forward and Reverse Analysis of Cellular Processes
24.5 Protein Selection Using mRNA Display

25 Discovery of Differentially Expressed Gene
25A.1 Laser Capture Microdissection
25A.2 Preparation of Single Cells from Solid Tissues for Analysis by PCR
25B.1 Production of a Subtracted cDNA Library
25B.2 PCR-Based Subtractive cDNA Cloning
25B.3 Differential Display of mRNA by PCR
25B.4 Restriction-Mediated Differential Display
25B.5 AFLP-Based Transcript Profiling
25B.6 Serial Analysis of Gene Expression (SAGE)
25B.7 Representational Difference Analysis
25B.8 Gene Expression Analysis of a Single or Few Cells

26 Gene Silencing
26.1 Overview of RNA Interference and Related Processes
26.2 Gene Silencing by RNAi in Mammalian Cells
26.3 RNA Interference in Caenorhabditis Elegans
26.4 Cloning of Small RNA Molecules (New, October 2003)
26.5 RNA Interference in Cultured <MI>Drosophila Cells (New, January 2004)

27 RNA-Protein Interactions
27.1 Agarose Gel Separation/Isolation of RNA-Protein Complexes
27.2 Identification of RNA Binding Proteins by UV Cross-Linking
27.3 Purification of Functional RNA-Protein Complexes Using MS2-MBP

Appendices
A.1 Standard Measurements, Data, and Abbreviations
A.2 Commonly Used Reagents and Equipment
A.3 Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots
A.4 Suppliers of Reagents and Equipment
A.5 Vectors

Index