PCR Protocols (Methods in Molecular Biology)

✍ Scribed by Daniel J. Park

• Publisher: Humana Press
• Year: 2010
• Tongue: English
• Leaves: 338
• Edition: 3rd ed.
• Category: Library

✦ Synopsis

Known for flexibility and robustness, PCR techniques continue to improve through numerous developments, including the identification of thermostable DNA polymerases which exhibit a range of properties to suit given applications. PCR Protocols, Third Edition selects recently developed tools and tricks, contributed by field-leading authors, for the significant value that they add to more generally established methods. Along with the cutting-edge methodologies, this volume describes many core applications, such as PCR cloning and sequencing, expression, copy number or methylation profile analysis, ‘DNA fingerprinting’, diagnostics, protein engineering, interaction screening as well as a chapter highlighting workflow considerations and contamination control, crucial for all PCR methods. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary reagents and materials, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, PCR Protocols, Third Edition seeks to further elucidate this essential technique while also providing core principles with broad applications for scientists of all backgrounds.

✦ Table of Contents

1.pdf......Page 1
PCR Protocols......Page 3
Preface......Page 5
Contents......Page 7
Contributors......Page 9
1. Introduction......Page 13
2. Materials......Page 14
3. Methods......Page 17
4. Notes......Page 22
References......Page 24
1. Introduction......Page 25
2. Materials......Page 27
3.2. PCR Cycling......Page 28
4. Notes......Page 29
References......Page 31
1. Introduction......Page 32
1.2. SplinkTA-PCR......Page 33
1.3. SplinkBlunt-PCR......Page 34
2.1. Extraction of Genomic DNA from Cell Culture or Tissues......Page 37
2.2. STA-PCR (Including DNA Digestion, Extension, Ligation, PCR Amplification, and Examination)......Page 38
3.1. Extraction of Genomic DNA from Cell Culture or Tissues......Page 39
3.2. STA-PCR (Including DNA Digestion, Addition, Ligation, PCR Amplification, and Examination) (see Notes 8–10)......Page 40
3.3. SplinkBlunt-PCR (Including DNA Digestion, Extension, Ligation, PCR Amplification, and Examination) (See Notes 9 and......Page 42
3.4. PCR Product Cloning and Sequencing......Page 44
4. Notes......Page 45
References......Page 48
1. Introduction......Page 50
2.1. 3 ¢RACE LaNe......Page 51
2.2.2. Terminal Transferase-Mediated 5¢RACE LaNe (TT RACE LaNe)......Page 53
2.3. LaNe RAGE......Page 54
3.1. 3¢RACE LaNe......Page 55
3.2.1. FI RACE LaNe......Page 56
3.2.2. TT RACE LaNe......Page 57
4. Notes......Page 58
References......Page 62
1. Introduction......Page 63
2.2. Template DNA Preparation......Page 66
3. Methods......Page 67
3.1. Primer Design: Manual Method......Page 68
3.2. Primer Design: Using iCODEHOP Software......Page 71
3.3. Template DNA Preparation......Page 73
3.4. CODEHOP PCR Amplification......Page 74
3.5. Sequence Analysis of PCR Product......Page 75
4. Notes......Page 76
References......Page 78
1. Introduction......Page 80
1.2. In Vitro Genomic Mutation......Page 81
1.3. Megabase Regions......Page 83
2.2. Mutagenic Nucleotide Triphosphates......Page 84
3.2.1. Reaction Conditions......Page 85
3.4.2. Use of Pairs of Mutagens......Page 86
3.5.1. Reaction Conditions......Page 87
3.8.1. Sequencing of Mutagenized Clones......Page 88
4. Notes......Page 89
References......Page 91
1. Introduction......Page 94
2.1. Single Cell Isolation......Page 95
2.2. cDNA Synthesis and Amplification......Page 96
2.6. Real-Time PCR......Page 97
3.1. Single Cell Isolation......Page 98
3.2. cDNA Synthesis and Amplification......Page 100
3.3. Template Preparation for IVT Labeling......Page 103
3.4. Additional Amplification (Optional)......Page 105
3.6. Spike RNA (Optional)......Page 106
3.7. Real-Time PCR (Optional)......Page 107
4. Notes......Page 108
References......Page 113
1. Introduction......Page 115
2.2.1. Stem-Loop RT......Page 117
2.2.4. Real-Time qPCR......Page 118
3.1.1. Total RNA......Page 119
3.1.2. Cell Lysate......Page 120
3.1.4. FFPE Samples......Page 121
3.2.1. Stem-Loop RT......Page 122
3.2.2. Real-Time qPCR......Page 123
3.3.1. Megaplex RT......Page 124
3.3.3. Real-Time qPCR......Page 125
3.4.1. Direct RT......Page 126
3.4.2.1. Poly(A) Tailing......Page 127
3.4.2.3. qPCR......Page 128
3.5.1. Selection of Endogenous Controls......Page 129
3.5.2. Data Normalization......Page 130
3.5.4. Clustering Analysis......Page 131
4. Notes......Page 132
References......Page 134
1. Introduction......Page 137
2.2. cDNA Synthesis......Page 139
2.4. Indexed-Adapter Ligation and PCR......Page 140
3. Methods......Page 141
3.3. Tag Extraction......Page 142
3.5. Purification of PCR Product......Page 144
4. Notes......Page 146
References......Page 148
1. Introduction......Page 149
2.1. Common, Reagents, Buffer, Equipment......Page 151
2.4. Wash Buffer......Page 152
2.13. Oligonucleotides......Page 153
3.1.3. GFX-CTAB Purification......Page 154
3.1.4. Biotinylation......Page 155
3.1.6. Cap-Trapping with Streptavidin-Sepharose Beads......Page 156
3.2.1. Single-Strand Linker Ligation......Page 157
3.2.2. GFX-CTAB Purification......Page 158
3.2.5. GFX-CTAB Purification......Page 159
3.3.3. Purification from the 3 ¢ Linker with Beads......Page 160
3.4.1. First PCR Check......Page 161
3.4.4. Purification and Sequencing......Page 162
4. Notes......Page 164
References......Page 165
1. Introduction......Page 166
2.1. Human Genomic DNA......Page 167
2.4. Emulsion Constituents......Page 168
3. Methods......Page 169
3.1. Primer Design......Page 170
3.2. Emulsion Formation......Page 171
3.5. Removal of Biotinylated Primers and Unlinked Amplicons......Page 172
3.7. Determination of Haplotypes by qASPCR......Page 173
3.8. Data Analysis......Page 174
References......Page 175
1. Introduction......Page 177
1.1. Overview of the PAP-LMPCR Procedure......Page 178
2.1. Primer Extension......Page 179
2.2. Product Capture......Page 180
2.5. Radioactive Labelling......Page 181
3.1.1. Primers......Page 182
3.1.4. Ligation......Page 183
3.1.6. Labelling of Reaction Products......Page 184
3.1.7. Electrophoresis of Radiolabelled DNA on a Polyacrylamide DNA-Sequencing Gel......Page 185
3.2. Allele-Specific PAP-LMPCR......Page 186
3.3. Representative Examples of In Vivo Footprinting Experiments Involving PAP-LMPCR......Page 187
4. Notes......Page 188
References......Page 191
1. Introduction......Page 193
2.1. Basic MLPA Reaction......Page 197
3.1. Basic MLPA Protocol......Page 198
3.3. Methylation Specific MLPA (MS-MLPA)......Page 201
3.5. DNaseI-MLPA......Page 202
4. Notes......Page 203
References......Page 204
1. Introduction......Page 206
3.1.1. Primer Design for Evaluation of Methylation Levels After Sodium Bisulphite Modification......Page 208
3.1.2. Optimisation
of Primer Annealing Temperature......Page 209
3.3. Setting up a Standard Curve of “Percentage of Methylation”......Page 211
4. Notes......Page 212
References......Page 215
1. Introduction......Page 217
2.3. Capillary Electrophoresis......Page 219
3.3. Capillary Electrophoresis......Page 220
3.4. Peak Analysis......Page 221
4. Notes......Page 222
References......Page 225
1. Introduction......Page 226
2.1. Asynchronous PCR Primer and Fluorogenic Probe Sequences (Examples)......Page 227
2.3. Asynchronous PCR for TaqMan® Assays......Page 229
3.1.2. PCR Protocol......Page 230
3.2.2. aPCR Protocol (Fig. 2)......Page 231
3.3. Generation of Single-Strand DNA Targets for Microarrays......Page 232
3.3.1. DNA Amplification and Labeling......Page 233
3.3.2. Purification of PCR Products......Page 234
4. Notes......Page 235
References......Page 237
1.1. The Limitations of Traditional PCR......Page 239
1.2. An Alternative Approach......Page 240
2.2. Use of Nucleic Acid Substrate for the Detection of ATP......Page 241
3.1. Use of Nucleic Acid Substrate for the Detection of Alkaline Phosphatase......Page 242
3.2. Use of Nucleic Acid Substrate for the Detection of ATP......Page 243
3.2.3. Detection of ATP......Page 244
3.3. Use of the ATP Assay for Antibiotic Susceptibility Testing of Cultured Bacteria......Page 245
3.4. Use of Nucleic Acid Substrate for the Detection of Bacterial Ligase......Page 246
3.4.2. Testing of Platelets for Bacterial Contamination......Page 247
4. Notes......Page 249
References......Page 250
1. Introduction......Page 251
2. Materials......Page 253
3.2. Thermocycling......Page 254
4. Notes......Page 255
References......Page 258
1. Introduction......Page 259
2.1.1. Standard DNA Oligos Modified to Not Prime Amplification......Page 260
3.1.1. Different Types of Blocking Principles......Page 261
3.1.2. Primer Designing......Page 262
3.2. Initial Tests of Blocking Oligo Performance and Optimisation of PCR Conditions......Page 263
3.4. Examples of PCRs Run with and Without Blocking Oligo......Page 264
References......Page 266
1. Introduction......Page 269
3. Methods......Page 271
4. Notes......Page 273
References......Page 274
1. Introduction......Page 275
2.1. General......Page 278
2.3. Reagents for mRNA Cleanup After In Vitro Transcription......Page 279
2.5. Reagents for In Vitro Transcription......Page 280
2.6.2. PremixZ......Page 281
2.7. Reagents for DARPin Expression and Binding Analysis of Single Clones (25)......Page 282
3.2. Transcription of PCR Products......Page 283
3.3. Cleanup of Template mRNA for In Vitro Translation......Page 284
3.4. In Vitro Translation......Page 285
3.5.2. Selection in Plates......Page 286
3.5.4. Affinity Maturation by Competition with Non-labeled Target (Off-Rate Selection)......Page 287
3.7. Reverse Transcription of DARPin mRNA......Page 288
3.10. Error-Prone PCR......Page 289
3.11. Initial Analysis of Selected Individual Library Members in a 96-Well Format (22)......Page 290
3.12. Preparation of S30 Extract (30–32)......Page 291
4. Notes......Page 292
References......Page 297
1. Introduction......Page 299
2.1. Oligonucleotides......Page 300
2.3. Cell-Free Protein Synthesis......Page 301
2.6. Sequencing Analysis......Page 302
3.1. Construction of GLOBE......Page 303
3.4. Recovery of DNA from GLOBE......Page 305
3.6. Gel-Mobility Shift Assay Linked with In Vitro Transcription/Translation......Page 306
4. Notes......Page 307
References......Page 309
1. Introduction......Page 310
1.2. TF Activities in Adult Mouse Tissues......Page 311
1.3. Correlation of MOUSE OATFA Tissue-Specific TF Activity (Cluster 3) with Gene Expression Activities......Page 313
2.1. Reagents......Page 314
2.3. Probe Sets......Page 315
3.2. OATFA Analysis of TF Activities......Page 316
3.4. Contrasting the Measured TF Activities......Page 318
4. Notes......Page 319
References......Page 321
1. Introduction......Page 323
2.1. Uridine-Exchange PCR......Page 325
2.5. Cloning......Page 326
3.1. NExTProg......Page 327
3.2. Uridine-Exchange PCR......Page 328
3.3. Enzymatic Digest and Chemical Cleavage......Page 329
3.4. Fragment Purification......Page 330
3.6. Cloning......Page 331
4. Notes......Page 332
References......Page 334
Index......Page 335