The Nucleic Acid Protocols Handbook


A comprehensive treasury of all the key molecular biology methods—ranging from DNA extraction to gene localization in situ—needed to function effectively in the modern laboratory. Each of the 120 highly successful techniques follows the format of the much acclaimed Methods in Molecular Biology™ series, providing an introduction to the scientific basis of each technique, a complete listing of all the necessary materials and reagents, and clear step-by-step instruction to permit ...
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A comprehensive treasury of all the key molecular biology methods—ranging from DNA extraction to gene localization in situ—needed to function effectively in the modern laboratory. Each of the 120 highly successful techniques follows the format of the much acclaimed Methods in Molecular Biology™ series, providing an introduction to the scientific basis of each technique, a complete listing of all the necessary materials and reagents, and clear step-by-step instruction to permit error-free execution.

Included for each technique are notes about pitfalls to avoid, troubleshooting tips, alternate methods, and explanations of the reasons for certain steps—all key elements contributing significantly to success or failure in the lab.

The Nucleic Acid Protocols Handbook constitutes today’s most comprehensive collection of all the key classic and cutting-edge techniques for the successful isolation, analysis, and manipulation of nucleic acids by both experienced researchers and those new to the field.

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Editorial Reviews

From the Publisher
"... the techniques are presented in minute details and in a "cookbook-standardized manner"... The personal notes inserted by each contributor provide assurance that the techniques would work in any laboratory... Having all of those prools updated and tested in practice and bound in one volume will be attractive to many scientists who deal with molecular biology techniques."- Modern Pathology

"There were over 190 authors involved in writing the prools, ...each prool is presented in outline format an follows a common numbering scheme, making it easy to find relevant sections....Many of the prools include tables of data or figures, including drawings or photographs of gels...One of the most useful sections in each prool lists notes, which elucidate possible pitfalls, provide troubleshooting tips, or give alternate methods or explanations for steps. This very practical information makes successful duplication of the procedure much more likely than when attempting to follow the often less detailed or less flexible descriptions given in journal articles....this handbook would be extremely useful as a reference in most university libraries."-E-Streams

" covers practically all of the very important and large field of nucleic acid research. The volume summarizes, in 10 parts, recent advances in the isolation , analysis and manipulation of nucleic acids. ...The chapters were contributed by authors from leading laboratories in the field all over the world....The book provides the reader with a large set of ready-to use prools needed to undertake complex molecular biology experimental project on a large spectrum of biological models involving isolation and analysis of nucleic acids. It is thus an almost indispensable handbook for anybody working in the field of molecular biology and genetics."-Folia Microbiologica

"...extremely useful as a reference in most university libraries." - E-Streams

"...comprehensive and detailed...this is an set of recipes for the genetic kitchen." -The Biologist

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Product Details

  • ISBN-13: 9780896034594
  • Publisher: Springer-Verlag New York, LLC
  • Publication date: 3/24/2000
  • Series: Methods in Molecular Biology Series
  • Edition description: 2000
  • Edition number: 1
  • Pages: 1072
  • Product dimensions: 7.00 (w) x 10.00 (h) x 2.11 (d)

Table of Contents

Preface v
Contributors xv
Part I Nucleic Acid Extraction
1 Isolation of High-Molecular-Weight DNA from Animal Cells 3
2 Isolation of mRNA by Affinity Chromatography 9
3 Isolation and Purification of DNA from Plants 13
4 Purification of Uncontaminated, Intact Plant RNA 17
5 An Improved Method to Isolate Mitochondrial RNA from Green Plant Tissue 23
6 Isolating Chromosomal DNA from Bacteria 29
7 Bacterial DNA Extraction for Polymerase Chain Reaction and Pulsed-Field Gel Electrophoresis 33
8 Isolation of Fungal Nucleic Acids 37
9 Total RNA Isolation from Bacteria 47
10 Simultaneous RNA and DNA Extraction from Biopsy Material, Culture Cells, Plants, and Bacteria 53
11 Spectrophotometric Analysis of Nucleic Acids 57
Part II Basic Separation and Analysis of DNA
12 Restriction Endonuclease Digestion of DNA 63
13 Agarose Gel Electrophoresis of Nucleic Acids 67
14 Preparation of RNA Dot Blots 71
15 Native Polyacrylamide Gel Electrophoresis 73
16 Southern Blotting of Agarose Gels by Capillary Transfer 77
17 Pulsed-Field Gel Electrophoresis 81
18 HPLC of DNA and PCR Products 105
Part III Probe Design, Synthesis, and Labeling
19 End-Labeling of DNA Fragments 117
20 Nick Translation and Random Hexamer Labeling of DNA 123
21 Generation of Labeled Probes by Polymerase Chain Reaction 127
22 Nonradioactive Oligonucleotide Probe Labeling 135
23 Preparation of Direct, Enzyme-Labeled DNA Probes 145
24 Random Prime Labeling of DNA Probes with Fluorescein-Tagged Nucleotides 149
25 Hybridization and Detection of Fluorescein-Labeled DNA Probes Using Chemiluminescence 153
26 Hybridization of Enzyme-Labeled Probes and Detection by Chemiluminescence 157
27 Hybridization and Competition Hybridization of Southern Blots 163
28 Autoradiography and Fluorography 169
Part IV RNA Analysis Techniques
29 Formaldehyde Gel Electrophoresis of Total RNA 177
30 RNA Probes for the Analysis of Gene Expression 181
31 Primer Extension Analysis of mRNA 195
32 S1 Mapping Using Single-Stranded DNA Probes 201
33 Measurements of Rate of Transcription in Isolated Nuclei by Nuclear "Run-Off" Assay 207
34 One-Tube RT-PCR with Sequence-Specific RT Primers 213
35 Characterization of RNA Using Continuous RT-PCR Coupled with ELOSA 219
36 Quantitative Analysis of RNA Species by Polymerase Chain Reaction and Solid-Phase Minisequencing 229
37 Nonradioactive Northern Blotting of RNA 239
38 Analysis of RNA by Northern Blotting Using Riboprobes 249
Part V Gene Library Construction and Screening
39 Production of Double-Stranded cDNA for Gene Library Synthesis 261
40 Using Rapid Amplification of cDNA Ends (RACE) to Obtain Full-Length cDNAs 267
41 cDNA Library Construction Using Streptavidin-Paramagnetic Beads and PCR 289
42 Rapid (Ligase-Free) Subcloning of Polymerase Chain Reaction Products 295
43 Subtraction Hybridization cDNA Libraries 305
44 Cloning Polymerase Chain Reaction Products Utilizing the T/A Overhang and a Kit 319
45 Extraction and Purification of Plasmid DNA 327
46 Biotinylated Probes in Colony Hybridization 333
47 Cloning Long Polymerase Chain Reaction Products 339
48 Cloning DNA Fragments in M13 Vectors 347
49 cDNA Library Construction for the Lambda ZAP-Based Vectors 355
50 Expression and Preparation of Fusion Proteins from Recombinant gt11 Phages 367
51 Antibody Screening of Bacteriophage gt11 DNA Expression Libraries 373
52 Screening cDNA Libraries by Hybridization with Double-Stranded DNA Probes and Oligonucleotides 381
53 cDNA Library Screening with the Tetramethylammonium Chloride (TMAC) Technique Using Highly Degenerate Oligoonucleotide Probes 389
54 Screening Recombinant Libraries by Polymerase Chain Reaction 397
55 Construction and Screening of Cosmid Libraries 405
56 Generation of Large Insert YAC Libraries 415
57 YAC Library Storage and Transport 425
58 YAC Library Screening: Preparation of Hybridization Filters and Polymerase Chain Reaction Pools 431
59 YAC Library Screening: Hybridization and PCR-Based Screening Protocols 437
60 Phage-Display Libraries of Murine and Human Antibody Fab Fragments 449
Part VI DNA Sequencing
61 Preparation and Analysis of DNA Sequencing Gels 481
62 DNA Sequencing of Plasmids 489
63 Sequencing DNA Fragments Cloned into M13 and Phagemid Vectors 493
64 Direct cDNA Sequencing Using Sequential Linear/Asymmetric Polymerase Chain Reaction 499
65 Purification and Enzymatic Sequencing of Polymerase Chain Reaction Products 505
66 Direct Polymerase Chain Reaction Sequencing with Denaturants 515
67 Direct DNA Sequencing of Polymerase Chain Reaction Products Using Magnetic Beads 523
68 Polymerase Chain Reaction Cycle Sequencing with Degenerate Primers 533
69 Direct Automated Cycle Sequencing of Polymerase Chain Reaction Products 541
70 Affinity-Capture and Solid-Phase Sequencing of Biotinylated Polymerase Chain Reaction Products 547
71 DNA Sequencing by the Chemical Method 553
72 One-Step One-Lane Chemical Sequencing of DNA 557
Part VII Basic Polymerase Chain Reaction Methods
73 Polymerase Chain Reaction: Basic Principles and Routine Practice 569
74 Primer Selection and Design for Polymerase Chain Reaction 581
75 One-Step Optimization Using Touchdown and Stepdown Polymerase Chain Reaction 589
76 Cloning Gene Family Members Using Polymerase Chain Reaction with Degenerate Oligonucleotide Primers 595
77 Construction of Synthetic Genes by Polymerase Chain Reaction 609
78 Rapid Amplification of cDNA Ends 613
79 Multiplex Polymerase Chain Reaction 619
80 Inverse Polymerase Chain Reaction 625
81 Long Range Polymerase Chain Reaction 633
Part VIII Analyzing Genes, Mutations, and Protein Interactions
82 Nonradioactive Differential Display of Messenger RNA 645
83 Gene Isolation by Exon Trapping 653
84 DNA Rescue by the Vectorette Method 667
85 Random Amplified Polymorphic DNA (RAPDs) 675
86 Restriction Fragment Length Polymorphism 679
87 Detection of Mutations in DNA and RNA by Chemical Cleavage 685
88 Mutation Screening Using PCR-SSCP: Silver Staining and Isotopic Protocols 695
89 Detecting Point Mutations by Denaturing-Gradient Gel Electrophoresis 705
90 Analysis of Nucleotide Sequence Variation by Solid-Phase Minisequencing 717
91 The Amplification Refractory Mutation System 723
92 DNase I Footprinting 729
93 Identification of Protein--DNA Contacts with Dimethyl Sulfate: Methylation Protection and Methylation Interference 737
94 The Gel Shift Assay for the Anmalysis of DNA--Protein Interactions 745
95 Yeast Two-Hybrid Library Screening 757
96 The Southwestern Assay 773
97 Nonradioactive Methods for the Detection of RNA-Protien Interaction 783
98 Tanscriptional Activation Analysis by the Chloramphenicol Acetyl Transferase (CAT) Enzyme Assay 793
Part IX Mutagenesis, Transcription, and Translation In Vitro
99 Generating Nested Deletions with Exonuclease III 807
100 Primer-Directed Site-Specific Mutagenesis 815
101 Site-Directed Mutagenesis Using a Uracil-Containing Phagemid Template 827
102 Site-Directed Mutagenesis Using Double-Stranded Plasmid DNA Templates 835
103 Site-Directed Mutagenesis with LA-PCR Technology 845
104 Recombination and Mutagenesis by Overlap Extension PCR 857
105 Site-Directed Mutagenesis and Gene Fusion by Megaprimer PCR 865
106 Transcription In Vitro Using Bacteriophage RNA Polymerases 875
107 In Vitro Translation of mRNA in a Rabbit Reticuloctye Lysate Cell-Free System 885
108 In Vitro Translation of mRNA in a Wheat Germ Extract Cell-Free System 891
109 The Xenopus Egg Extract Translation System 895
110 Manipulation of Baculovirus Vectors 907
111 Procedures for the Analysis and Purification of His-Tagged Proteins 921
112 Detection and Immobilization of Proteins Containing the 6xHis Tag 935
113 Expression and Purification of Recombinant Proteins Using the pET System 947
Part X Gene Localization, Mapping In Situ, and Bioinformatics
114 Preparation of Tissue Sections and Slides for mRNA Hybridization 981
115 Use of Digoxigenin-Labeled Probes on Tissue Sections 985
116 Gene Mapping by Fish 991
117 Oligonucleotide Prins DNA Synthesis 1011
118 Chromosome-Specific Prins 1017
119 In Situ PCR Amplification of Intracellular mRNA 1023
120 An Introduction to Bioinformatics 1031
Index 1045
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