Nonmammalian Genomic Analysis: A Practical Guide

✍ Scribed by Bruce Birren, Eric Lai

• Publisher: Academic Press
• Year: 1996
• Tongue: English
• Leaves: 369
• Category: Library

✦ Synopsis

Offering detailed protocols for those needing to construct a variety of maps and isolate genes, this unique book is intended to popularize the new techniques of genome analysis derived from the Human Genome Project. The power of these new methods is often most striking when applied to problems outside of human genetics, particularly the nonmammalian systems on which many researchers focus. Many of these organisms are economically important and biologically rich.Nonmammalian Genomic Analysis: A Practical Guide covers the "how to" aspects of preparation, handling, cloning, and analysis of large DNA and the creation of chromosome and genome maps. This lab manual facilitates the transfer of these technologies to small "low tech" environments and allows them to be used by those with no background in genome mapping or large-fragment cloning. Like having a local expert, this collection provides procedures for anyone, anywhere, and allows the replication of others' success. Key Features Includes detailed and clearly-written step-by-step protocols Evinces expected results and offers trouble shooting advice Provides techniques appropriate for small laboratories as well as those with limited resources Covers a broad variety of cloning systems, including single copy vectors* Discusses a diverse range of organisms, from prokaryotes to eukaryotes, from single-celled organisms to highly complex organisms

✦ Table of Contents

Cover
......Page 1
Title Page
......Page 4
Copyright......Page 5
Contents......Page 6
Preface......Page 14
A. Introduction......Page 16
C. Switch Time Governs PFG Resolution......Page 17
D. Establishing Effective Separation Conditions......Page 21
E. Selection of a PFGE Instrument
......Page 25
A. Electrophoresis Buffers......Page 26
B. Solutions......Page 27
A. General Principles......Page 28
1. Preparation of Yeast Chromosomes by Embedding Intact
Cells in Agarose
......Page 30
2. Preparafion of Chromosomal DNA from Bacteria......Page 31
C. Controlling and Determining DNA Concentration......Page 32
A. General Principles......Page 33
V. Southern Blotting of Pulsed-Field Gels......Page 34
VI. Troubleshooting Pulsed-Field Gels......Page 37
References......Page 38
I. Introduction......Page 40
II. Choice of Sample Material......Page 44
III. Sample Preparation......Page 45
A. Materials......Page 46
1. Saccharomyces cerevisiae and Other Fungi......Page 49
2. Neurospora crassa
and Other Fungi......Page 50
3. Chopped Inserts and Agarose Bead Encapsulation......Page 52
B. Constructing Electrophoretic Karyotypes......Page 53
1. Determining ChDNA Quality......Page 54
2. Determining Chromosome Size Range and Distribution......Page 57
3. Optimizing Electrophoretic Karyotypes......Page 58
V. Applications of Electrophoretic Karyotyping......Page 62
VI. Conclusion......Page 67
References......Page 68
I. Introduction......Page 76
IL Materials......Page 77
A. Isolation of High-Molecular-Weight DNA from Protoplasts
of Tomato Leaves
......Page 78
1. PreparaHon of Agarose Blocks......Page 79
B. Procedure for Restriction Enzyme Digestions......Page 80
A. Protoplast Quality and Isolation......Page 81
2. Protoplast Isolation......Page 82
B. Other Useful Hints for Protoplast Isolation and Embedding
in Agarose
......Page 83
3. Digestion of DNA in Agarose......Page 84
E. Isolation of High-Molecular-Weight DNA from Other Plants......Page 86
References......Page 87
I. Introduction......Page 90
II. Comparison of DNA Marker Systems......Page 91
A. Information Content......Page 92
III. Materials......Page 94
IV. Restriction Fragment Length Polymorphism Markers......Page 95
A. Probe Considerations......Page 96
B. Mapping Populations......Page 97
C. Polymorphism Screen......Page 98
D. Progeny Genotyping......Page 99
F. Experimental Considerations: Materials......Page 100
V. Cleavable Amplified Polymorphic Sequences......Page 102
A. Experimental Considerations......Page 103
B. ReproducibiUty of RAPDs......Page 104
D. Near Isogenic Lines (NILs)......Page 105
E. Pooling Strategies......Page 106
G. Experimental Protocol: Materials......Page 108
Vll. Microsatellite Markers (Simple Sequence Repeats, SSR)......Page 111
A. Materials......Page 113
B. Genomic DNA Library Construction......Page 114
C. Library Screening by Hybridization......Page 116
D. DNA Sequence Determination of Positive Clones......Page 118
F. PCR Analysis and Identification of Polymorphisms......Page 121
G. Polymorphic Marker Mapping......Page 122
VIIL Sequence-Based Polymorphism Assays......Page 123
B. Allele-Specific Ligation......Page 124
IX. Higher Multiplex Ratio Assays: Amplified Fragment
Length Polymorphism and Interrepeat Amplification
......Page 125
A. Amplified Fragment Length Polymorphism......Page 126
1. Experimental Method: Materials......Page 128
2. Advantages and Other Considerations......Page 136
B . Interrepeat Amplifications......Page 138
X. choosing Appropriate Technology......Page 142
Acknowledgments......Page 143
References......Page 144
I. Introduction......Page 150
IL Strategy......Page 151
B. Buffers......Page 153
1. Theileria and Babesia Species......Page 156
2. Trypanosoma species......Page 157
B. Preparation of Agarose-Embedded HMW DNA......Page 158
C. Identification of Appropriate Rare-Cutting Restriction Enzymes......Page 159
2. Separation of Sfil Fragments of Theileria and Babesia Species......Page 160
4. Separation of Trypanosoma Chromosomes......Page 161
1. Southern Blotting......Page 162
E. Identification
of Telomeres......Page 163
1. Partial Digestion of Theileria parva Genomic DNA by Sau3AI......Page 164
3. Ligation of Partially Filled Vector and Target DNAs......Page 166
4. Transformation of Escherichia coli Strain JM83......Page 167
5. Enrichment of Linking Clones......Page 168
1. Identification of Sfil Linking Clones......Page 169
2. Characterization of Sf~l Linking Clones......Page 170
H. Ordering of Macrorestriction Fragments Using Linking Clones
as Probes
......Page 172
I. Completion and Confirmation of tiie Map Using
Otiier Techniques
......Page 175
V. General Remarks and Conclusions......Page 176
References......Page 177
II. Materials......Page 180
2. Dpn\ Buffer......Page 181
F. MgCl2 (1 M)......Page 182
M. lOX TE......Page 183
III. Topology of Bacterial Genomes......Page 184
A. Protocols for the Determination of the Number and Size
of Replicons
......Page 186
A. Choice of Restriction Endonucleases......Page 188
B. Identification of Fragment Number......Page 189
C. Mapping Strategies......Page 191
D. Two-Dimensional Mapping......Page 193
References......Page 209
I. Introduction......Page 212
II. General Considerations......Page 213
III. Choice of Vector......Page 214
1. Material......Page 217
2. Procedure......Page 218
1. Procedure: Preparation of Partially Digested Genomic DNA......Page 220
D. In Vitro Packaging......Page 222
E. Infection......Page 223
R Amplification and Storage......Page 224
A. Comparison of Clones by Restriction Analysis......Page 225
B. Chromosome Walking......Page 230
VL Mapping Problems......Page 232
Procedure for Linking Adjacent Cosmid......Page 233
VIL Summary......Page 234
References......Page 235
I. Introduction......Page 238
A. Supply Checklist for PAC Cloning......Page 241
B. Stock Solutions......Page 242
A. General Precautions......Page 244
B. Preparation of PAC Vector for Cloning......Page 245
C. Embedding of Erythrocytes in Agarose Blocks for High-
Molecular-Weight DNA
......Page 249
D. Partial Restriction Digests and Size-Selection of DNA......Page 251
E. Generation of PAC Clones......Page 257
F. Library Construction and Screening......Page 259
G. Rapid DNA Isolation from PAC Clones......Page 261
H. Restriction Mapping of PAC Clones......Page 262
IV. Summary......Page 267
References......Page 268
I. Introduction......Page 272
IL Materials......Page 273
A. Creating Genomic Libraries of Trypanosome DNA......Page 276
2. Bacteriophage PI......Page 277
1. Pulsed-Field Gel Electrophoresis......Page 278
2. Elution of fhe Chromosome and Preparation of a Complex Probe......Page 279
C. Preparation of Colony Lifts and Hybridization Conditions......Page 281
1. Preparation of Replica Filters by Random Plating......Page 282
2. Preparation of an Arrayed PI Library and Replica High-Density
Filters
......Page 284

3. Hybridization of Gel-Purified DNA to Cosmid and PI Filters......Page 285
2. Storing fhe Chromosome-Specific Clones......Page 293
1. Hybridization to Pulsed-Field Gels......Page 294
2. Restriction Digests of Cosmid and P1 Clones......Page 296
1. Creating Contigs......Page 298
2. Selecting Chromosome-Specific Markers from the Subgenomic
Library
......Page 299
H. Summary of Results......Page 300
A. Low Levels of Hybridization......Page 301
D. Gel-Eluted Probes May Be Amplified......Page 302
F. Use Your Knowledge about the Repeated DNA Content
of the Genome
......Page 303
Reference......Page 304
A. Overview......Page 308
B. Genetics and Physical Genome Analysis......Page 309
A. Growth Media for Dictyostelium......Page 310
B. Solutions......Page 312
A. Preparation of High-Molecular-Weight Nuclear DNA......Page 313
B. Digestion of DNA Embedded in Agarose......Page 314
IV. The Use of YACs in Genome Analysis......Page 315
A. YAC Library Construction......Page 316
B. YAC Contig Construction......Page 319
C. Characterization of Gene Families Using YACs......Page 320
D. Subcloning Dictyostelium Genes from YACs......Page 321
V. Restriction Enzyme-Mediated Integration
(REMI)-RFLP Analysis
......Page 322
A. Construction of REMI-RFLP Strains
......Page 323
B. REMI Transformation Protocol......Page 324
C. REMI-RFLP Analysis......Page 326
VI. Random Insertional Mutagenesis Using REMI......Page 328
A. KEMI Transformation and Mutant Screening......Page 329
B. Cloning Sequences Flanking Insertion Sites......Page 330
References......Page 331
I. Introduction......Page 334
A. Solutions and Reagents......Page 336
A. Tools for Hybridization Analyses......Page 338
B. Hybridization Techniques......Page 345
C. Map Construction......Page 350
D. Data Handling......Page 357
IV. Conclusions......Page 358
References......Page 359
Index......Page 362