Genomics Protocols

1.1 Multiple Displacement Amplification and Other Methods for Whole Genome Amplification......Page 16
1.2 Applications in Which Whole Genome Amplified DNA Have Been Used......Page 17
1.3 Why Low Yield DNA Samples Should Be Studied......Page 20
2.2 Kits......Page 21
2.4 Additional Laboratory Reagents and Solutions......Page 24
3.2 Whole Genome Amplification......Page 25
3.4 Recommended Quality Checks......Page 26
3.4.3.1 Sex-PCR......Page 27
3.4.3.2 Quantitative PCR......Page 29
3.5 Pooling of WGA Products......Page 30
References......Page 31
E:\\Booksforhome\\genomics\\back-matter.pdf......Page 0
1 Introduction......Page 34
2.3 Allele separation......Page 35
3.2 PCR......Page 36
3.3 Denaturant Capillary Electrophoresis......Page 38
4 Notes......Page 39
References......Page 47
1 Introduction......Page 50
2.2 Setting up Sequencing Reactions......Page 52
3.1.1 Plasmid Preparation......Page 53
3.1.3 Quantities of Template DNA Required for Cycle Sequencing Reactions......Page 54
3.2 Setting up the PCR Reaction Using the Big Dye Cycle Sequencing Kit......Page 55
3.4 Sequencing on ABI DNA Analyzer......Page 56
3.5.1 Analysis Programs......Page 57
3.5.3 BLAST Analysis......Page 58
3.6.1 Overview......Page 59
3.6.3 Heterozygous (Double) Peaks......Page 61
3.6.7 Sequencing Artifacts......Page 62
4 Notes......Page 63
References......Page 66
1 Introduction......Page 68
2 Materials......Page 69
3.2 Sample Preparation......Page 71
3.3.2 Day 2. 1300 –1700 —Hybridization to Array (Overnight)......Page 72
3.4 Analysis of Custom Assays......Page 73
3.4.3 Review the DNA Quality and Select Samples for Exclusion......Page 74
3.4.5 Review and Edit Clusters......Page 76
3.4.6 Types of Clusters......Page 77
3.5.1 Inheritance Problems......Page 79
3.5.2 Poor Separation......Page 81
3.5.3 Assay-Specific Problems......Page 82
3.6 Strategy for Problem Solving......Page 84
3.7 Analysis of Standard Content Panels......Page 85
4 Notes......Page 87
References......Page 88
1 Introduction......Page 90
2.2.2 DNA Extraction from Buccal Swabs......Page 92
3.1 Selection of Candidate Genes......Page 93
3.2 Identification of Microsatellites Closely Flanking Candidate Gene(s)......Page 94
3.3 PCR Primer Design......Page 95
3.4.1 DNA Preparation from 2–10 ml of Blood Using a Nucleon Kit......Page 96
3.6 Fractionation of Fluorescently Labeled Microsatellite Alleles on a 3100 Genetic Analyzer......Page 97
3.8 Linkage and Association Analysis (see Note 6)......Page 98
4 Notes......Page 99
References......Page 100
1 Introduction......Page 102
2.3 DNA Isolation and Digestion......Page 103
2.5 Hybridization and Wash......Page 104
3.2 Genomic DNA Preparation......Page 105
3.3 Synthesis of Cy3- and Cy5-Labeled Targets......Page 106
3.4 Preparation of Labeled Target and Hybridization......Page 107
3.4.2 MAUI Hybridization Set......Page 108
3.6 Array Scanning......Page 109
3.7 First-Pass Analyses......Page 110
3.8 QA Assessment......Page 111
4 Notes......Page 113
References......Page 115
1 Introduction......Page 116
2.2 MLPA Reaction......Page 117
3.2 MLPA Reaction......Page 118
3.3.1 ABI-310 (One Capillary)......Page 119
3.4.2 Calculations of the Relative Probe Ratios......Page 120
4 Notes......Page 121
References......Page 122
1 Introduction......Page 124
2.1 Adapter Design......Page 125
2.6 Purification of Amplification Products......Page 127
3 Methods......Page 128
3.1 Adapter Design......Page 129
3.2 Methylation-Sensitive Cleavage of DNA......Page 130
3.3 Ligation......Page 131
3.5 CpG-Specific Adapter Amplification......Page 133
3.6 Purification of Amplification Product......Page 134
3.7.2 Quenching......Page 135
3.7.4 Determination of Incorporated Dyes......Page 136
3.8.2 Hybridization......Page 137
3.9 Examples......Page 138
4 Notes......Page 141
References......Page 144
1 Introduction......Page 145
2.1 Cell Culture and Cross-Linking......Page 146
2.3 Immunoprecipitation and Reverse Cross-Linking......Page 147
2.5 Microarray Hybridization and Scanning......Page 148
3.2 Cell Lysis, Preparation of Chromatin Complexes and Immunoprecipitation......Page 149
3.4 Purification, qPCR and Amplification of DNA......Page 150
3.5 Labeling, Hybridization, and Scanning......Page 151
3.6 Data Normalization and Peak Identification......Page 153
4 Notes......Page 155
References......Page 158
1 Introduction......Page 160
2.2 First-Strand cDNA Synthesis......Page 164
2.5 IVT for TC RNA Amplification: Biotinylation/Fluorescent Probe Labeling......Page 165
3.1 Isolation of RNA......Page 166
3.3 Second-Strand cDNA Synthesis......Page 167
3.6 IVT for TC RNA Amplification: Radioactive Probe Labeling......Page 168
4 Notes......Page 169
References......Page 170
1.1 Introduction to BeadArray Technology and Randomly Assembled Bead Arrays......Page 172
1.2 The DASL Assay for Gene Expression Profiling......Page 173
2.2 Illumina Supplied Reagents for the DASL Assay......Page 175
2.3 Other Reagents Required for the Assay......Page 176
2.5 Bead Array Imaging......Page 177
3.2 DASL Assay Protocols......Page 178
3.2.2.1 The Make SUR (Single Use RNA) Process for cDNA Synthesis......Page 179
3.2.2.3 The Add MEL Process for Assay Oligonucleotide Extension and Ligation......Page 180
3.2.2.5 The Make HYB Process to Prepare Samples for Array Hybridization......Page 181
3.3 Hybridization to Bead Arrays......Page 182
3.5 Data Collection and Analysis......Page 183
4 Notes......Page 184
References......Page 190
1 Introduction......Page 191
2.1 Macro- and Microarrays......Page 193
2.2 Probe Preparation......Page 195
2.3 Probe Amplification......Page 196
3 Identification of miRNA Target Genes......Page 198
References......Page 200
1 Introduction......Page 203
2 Material......Page 204
2.4 Binding Buffers for the Different Chip Arrays......Page 205
3.1 Sample Preparation for Storing......Page 206
3.5 Mass Analyzer......Page 207
4 Notes......Page 208
References......Page 209
1 Introduction......Page 210
2.3 Capture And detection of Cells on DotScan Microarrays......Page 212
3.1.1 Leukemias and Lymphomas from Peripheral Blood......Page 213
3.2.1 Leukaemia Cells......Page 214
3.3.2 Fluorescence Multiplexing......Page 215
3.6 Target Recognition for Therapeutic Antibodies......Page 217
4 Notes......Page 218
References......Page 219
1 Introduction......Page 221
2.1 Sample Preparation......Page 222
2.4 SDS-PAGE......Page 223
3.1 Sample Preparation......Page 224
3.2 Experimental Setup and Protein Labeling with Fluorescent Dyes......Page 225
3.3 Isoelectric Focusing......Page 226
3.4.1.1 Preparation of the Gel Caster......Page 227
3.4.3 SDS-PAGE......Page 228
3.5 Gel Imaging and Data Analysis......Page 229
3.6 Protein Identification......Page 231
4 Notes......Page 232
References......Page 234
1 Introduction......Page 235
2.1 Protein Samples......Page 236
2.3 Mass Spectrometry and Online Reverse-Phase Liquid Chromatography......Page 238
3 Methods......Page 239
3.1 Protein Sample Preparation......Page 240
3.3 ICAT Labeling and Tyrpsin Digestion of Samples......Page 241
3.4 Cleaning up and Fractionating Digested Samples Using the CE Cartridge......Page 242
3.6 NanoLC-MS and Data Analyses......Page 243
3.7 NanoLC-MS/MS Analysis of Differentially Expressed Peptides and Database Searching......Page 244
4 Notes......Page 247
References......Page 250
1 Introduction......Page 251
2.3 Mass Spectrometry and Online Reverse Phase Liquid Chromatography......Page 254
3.2 Reducing, Alkylating, and Trypsin Digesting the Samples......Page 255
3.3 Fractionating and Cleaning up Digested Samples Using a CE Cartridge......Page 256
3.5 MatchRx Software and Statistical Analysis......Page 257
3.6 NanoLC-MS/MS Analysis of Differentially Expressed Peptides and Database Searching......Page 263
4 Notes......Page 264
References......Page 266
1 Introduction......Page 267
2.2 Equipment and Reagents......Page 269
3.1 Preparation of the Peptide Extracts for 2D-LC-MS/MS Analysis......Page 270
3.3 2D-LC-MS/MS......Page 271
3.4 Database Searching Using Mascot and the Identification of F. tularensis Peptides......Page 272
3.5 Confirmation That the Putative F. tularensis Peptides Are Exclusively Expressed in the Infected Cell Line......Page 273
4 Notes......Page 274
References......Page 277
1 Introduction......Page 278
2.4 Plasticware......Page 281
2.6 General Stock Solutions and Media......Page 282
2.8 Buffers for Protein Purification......Page 283
3.1.1 Initiation of Cell Cultures......Page 284
3.2 Cloning the Gene of Interest......Page 285
3.3 High-Throughput Bacmid Production......Page 287
3.4 Confirmation of Transposition by PCR on Bacmid DNA......Page 288
3.5 Transfection and Infection of Insect Cells......Page 289
3.6 Plaque Purification......Page 291
3.8 High-Throughput Small-Scale Expression Study......Page 292
4 Notes......Page 294
References......Page 297
1 Introduction......Page 299
2.3 Coimmunoprecipitation......Page 302
2.6 Mass Spectrometry Compatible Silver Staining......Page 303
2.9 Nano-HPLC-Ion-Trap-MS/MS......Page 304
3.1 Cell Culture......Page 305
3.3 Coimmunoprecipitation......Page 306
3.4 SDS-PAGE......Page 307
3.6 Silver Staining......Page 308
3.8.1 Excising and Shrinking the Gel......Page 309
3.9 NanoHPLC-Ion-Trap-MS/MS......Page 310
4 Notes......Page 311
References......Page 316
1 Introduction......Page 317
2.1 Yeast Cell Culture and Lysis......Page 320
2.4 Purification After In-Vivo Cross-Linking Using the HBH Affinity Tag......Page 321
2.6 Sample Preparation for “In-Gel” Identification......Page 322
3.1.1 Growth and Lysis of Yeast Cells Expressing TAP-Tagged Proteins......Page 323
3.1.3 Tandem Affinity Purification Using ProtA/CBP-Tagged Bait Proteins......Page 324
3.2.1 Growth, In-Vivo Cross-Linking, and Lysis of Yeast Cells Expressing HB-Tagged Proteins......Page 326
3.2.3 Tandem-Affinity Purification of HBH-Tagged Proteins......Page 327
3.2.5 Western Blot Analysis of Purification......Page 328
3.3.2 Excision and “In-Gel” Digestion of Protein Bands......Page 329
3.3.3 Mass Spectrometry......Page 330
3.4.3 LC-MS/MS Analysis......Page 331
4 Notes......Page 332
References......Page 333
1 Introduction......Page 335
2.1 PCR and Cloning of Genes of Interest into Mammalian Two-Hybrid Vectors......Page 337
2.4 Transfection and Mammalian Two-Hybrid Analysis......Page 338
3.2.1 Restriction Digestion......Page 339
3.2.4 Screening for Clones That Contain the Gene of Interest......Page 340
3.4.1 Culturing and Preparing Vero E6 Cells......Page 341
3.5.1 SEAP Reaction......Page 342
4 Notes......Page 343
References......Page 344
1 Introduction......Page 346
2.1 Cloning and DNA Production......Page 348
2.4 Western Blot Materials......Page 349
3.1 DNA Construction and Manipulation into TriEx3Neo Expression Cassette......Page 350
3.2.1 Preparation of Mammalian Cell Lysates......Page 351
3.3.1 Preparation of Mammalian Cell Lysates for Western Blot Assay......Page 352
3.4.1 Mammalian Cell Transfection......Page 353
3.4.2 Imaging Live Mammalian Cells Using the Xenogen IVIS (In Vivo Imaging System)......Page 354
3.5.3 Pseudotumor Implantation......Page 355
3.6 In-Vivo Mouse Experiments Using Hydrodynamic Injections for Hepatocellular Somatic Gene Transfer......Page 356
3.6.2 Mouse Imaging......Page 357
References......Page 358
1 Introduction......Page 360
2.2 Cell Culture and Transfection......Page 362
3.1 Obtaining an Open Reading Frame Clone......Page 363
3.2.1 Choosing an Expression Vector......Page 364
3.2.2 Transferring an ORF into an Expression Vector......Page 365
3.3.1 Preparing DNA for Transfection......Page 367
3.4.1 Fixing and Mounting Cells......Page 368
3.4.2 Detecting Gene Epitope Tags with Antibody......Page 369
3.5 Performing a Western Blot......Page 370
3.6 Analysing the Results......Page 371
4 Notes......Page 372
References......Page 373
1 Introduction......Page 375
2.1.1 Database Component......Page 377
2.1.3 GUI Component......Page 380
3.1 Search by ORF Name, Gene Name, or Keyword......Page 381
3.2 Prediction of Function of the Gene of Interest......Page 386
3.3 Testing and Training Using Different Probability Functions and Datasets......Page 388
4 Notes......Page 391
References......Page 392
1 Introduction......Page 393
2.1 Databases......Page 395
2.2 Sequence Comparison......Page 396
2.4 Integrated Servers......Page 397
3.1 Detection of Orthology......Page 398
3.1.3 Phylogeny-Based Orthology Inference......Page 399
3.2.2 Conservation of Gene Order......Page 400
3.3 Phylogenetic Profiling......Page 401
3.4 Lineage-Specific Gene Loss......Page 402
3.5 Other Forms of Coevolution......Page 403
4 Notes......Page 404
References......Page 405
1 Introduction......Page 408
2.2 Lipid Mediated Oligonucleotide Transfection into Mammalian Cells......Page 411
2.4.2 SDS-PAGE and Western Blotting......Page 412
2.6 Invasion Assay......Page 413
3.2 Synthesis of Oligonucleotides (Synthesis, Purification, Enhancing Modifications)......Page 414
3.4 Methods of siRNA Delivery......Page 415
3.5.2.1 Western Analysis (Fig. 27.3)......Page 416
3.5.2.2 Immunofluorescent Staining (see Fig. 27.4)......Page 419
3.5.2.3 Functional Assays (see Fig. 27.5)......Page 420
4 Notes......Page 422
References......Page 423