The PCR Revolution

Half-title......Page 3
Title......Page 5
Copyright......Page 6
Contents......Page 7
Contributors......Page 9
Foreword......Page 13
References\n......Page 14
Preface......Page 15
References\n......Page 17
I Basic technologies......Page 21
1 Real-time polymerase chain reaction......Page 23
Early days......Page 24
The Story Unfolds......Page 25
Understanding the PCR Reaction......Page 26
Quantification Prospects......Page 27
Real-Time PCR......Page 29
References......Page 31
Thermostable enzymes......Page 32
PCR......Page 33
Use of thermostable polymerase enzymes for PCR......Page 34
Different thermostable enzymes used in PCR......Page 35
Future trends: Transgenic organisms for producing thermostable enzymes......Page 36
Conclusions......Page 37
References......Page 38
3 Inventing molecular beacons......Page 39
First real-time exponential amplification assays......Page 40
Smart probes containing molecular switches......Page 41
Misery loves company......Page 43
Probes that become fluorescent upon hybridization......Page 44
Contact quenching enables multicolor probes......Page 46
Real-time PCR assays with molecular beacons......Page 48
The extraordinary specificity of molecular beacons......Page 49
Molecular beacons as biosensors......Page 51
Observing the movement of mRNAs in live cells......Page 54
Practical applications......Page 56
References......Page 57
4 Rapid polymerase chain reaction and melting analysis......Page 68
Minimizing the time for PCR......Page 70
Commercial PCR instrumentation......Page 71
Microsystem PCR......Page 73
Ideal temperature profile for rapid-cycle PCR......Page 74
Denaturation......Page 75
Annealing......Page 76
Melting analysis......Page 77
PCR product melting......Page 78
High-resolution melting analysis......Page 79
Heterozygote scanning......Page 81
Genotyping by probe melting......Page 83
References......Page 85
Illuminating the unseen......Page 90
Isotopes and fluorophores......Page 91
Dna by design......Page 92
Success breeds failure......Page 94
Fluorogenic probes......Page 96
Transforming tomorrow......Page 97
References......Page 98
6 Analysis of microribonucleic acid expression by quantitative real-time polymerase chain reaction......Page 100
Detection of miRNA precursors by qPCR......Page 101
qPCR detection of mature miRNA via a synthetic poly(A) tail–mediated RT......Page 102
qPCR detection of mature miRNA via RT with stem–loop primers......Page 103
miRNA qPCR assays also require controls......Page 104
Conclusion......Page 105
References......Page 106
7 Miniaturized polymerase chain reaction for quantitative clinical diagnostics......Page 108
Current quantitative polymerase chain reaction uses in quantitative clinical diagnostics......Page 109
Applications......Page 110
Quality control......Page 111
cDNA synthesis......Page 112
PCR with fluorescent resonance energy transfer probes......Page 113
Normalization......Page 114
Can current technology achieve miniaturized quantitative clinical diagnostics?......Page 115
Siliconglass-based......Page 116
Chamber-based stationary PCR......Page 117
Shunting PCR......Page 118
qRT–PCR......Page 119
Constraints associated with mu–TAS for qPCR......Page 120
Detection......Page 121
Applications of quantitative clinical diagnostics......Page 122
Conclusions and perspectives......Page 123
References......Page 124
Introduction......Page 130
Pre-PCR steps......Page 131
Inventions made in “absolute” quantification assays......Page 132
How the relative quantification strategy changed......Page 134
What about PCR efficiency?......Page 135
Assay variance and how to perform a proper normalization......Page 136
Expression profiling, qPCR bioinformatics, and statistical analysis......Page 137
What is next in real-time PCR?......Page 138
Gene expression and more – the system biology idea......Page 140
References......Page 144
DNA analysis in the pre-PCR era......Page 149
PCR......Page 152
The modern PCR......Page 155
Quantitative PCR......Page 159
The PCR revolution......Page 161
Controlling the revolutionaries......Page 164
“With all its appropriate safeguards”......Page 165
References......Page 170
II Applications......Page 173
10 Polymerase chain reaction–based methods for the detection of solid tumor cancer cells for clinical diagnostic and prognostic assays......Page 175
The Clinical Need to Accurately Detect Metastatic Disease......Page 176
Conventional Methods and Limitations......Page 177
Amplification of tumor-specific abnormalities......Page 178
Amplification of tumor-associated wild-type mRNA......Page 179
TH mRNA in NBL......Page 180
PSA mRNA in prostate cancer......Page 181
Tyrosinase mRNA in malignant melanoma......Page 182
References......Page 184
11 Polymerase chain reaction and infectious diseases......Page 193
Infectious Disease Diagnosis (Jargon)......Page 194
Diagnostic Techniques......Page 195
Viruses......Page 196
Bacteria......Page 198
Genetic Relationships......Page 199
Why Is PCR Not Used More Routinely for Infectious Disease Diagnosis?......Page 201
Are molecular methods of any value for diagnosing diseases like TB?......Page 203
PCR for tropical disease diagnosis......Page 204
The role of PCR in the future of TB Diagnosis......Page 205
References......Page 206
12 Polymerase chain reaction and respiratory viruses......Page 209
A brief introduction to the features of respiratory viruses and the role of polymerase chain reaction in detecting them......Page 210
Why respiratory virus PCR is nothing to sneeze at......Page 212
PCR is the best tool we have…but not the perfect tool......Page 213
How do we make oligonucleotides into good primers?......Page 216
Therapies drive better diagnostics, but research prioritizes the targets for drug development......Page 217
Self-contained amplification means never having to say you contaminated your laboratory......Page 218
Conventional PCR was exceptionally sensitive; real-time PCR…is too......Page 219
Cost is absolutely relative......Page 220
Chemistry: will this be on the exam?......Page 221
qPCRs – Why most are not......Page 222
Identifying the emerging challenges of NIVs......Page 223
Raging infection or just gene detection?......Page 224
You say co-infection, I say codetection......Page 225
The KI and WU polyomaviruses......Page 226
HBoV......Page 227
Conclusions......Page 228
References......Page 229
13 Polymerase chain reaction and severe acute respiratory syndrome......Page 232
The taxonomy of the SARS coronavirus......Page 233
Structure and morphography of SARS-CoV......Page 234
Propagation of SARS-CoV......Page 251
SARS-CoVs genome......Page 252
Animal-to-animal transmission......Page 255
Diagnostic methods......Page 256
Laboratory methods for SARS diagnosis......Page 257
Legacy PCR assays......Page 259
Real-time PCR assays (qPCR)......Page 260
References......Page 262
14 The MMR vaccine, measles virus, and autism – A cautionary tale......Page 265
Measles virus......Page 267
Not just one problem, but one after another after another......Page 268
The final proof......Page 275
Conclusions......Page 276
References......Page 277
15 Noninvasive prenatal diagnosis using cell-free fetal nucleic acids in maternal plasma......Page 279
Discovery of cell-free fetal DNA in maternal plasma......Page 280
Methylation-specific PCR and related technologies: the development of fetal epigenetic markers......Page 281
Mass spectrometric analysis of PCR products from maternal plasma for trisomy 21 detection: in search of the “holy grail” of prenatal diagnosis......Page 283
Conclusions: PCR as a key facilitator in the past decade of progress......Page 285
References......Page 286
16 Polymerase chain reaction–based analyses of nucleic acids from archival material......Page 290
Advantages of archival specimens......Page 291
Reconstruction of a pandemic (Spanish flu)......Page 292
Molecular archaeology......Page 293
“Small” means “archival”......Page 294
Analysis of archival material in daily practice......Page 295
Suggested reading......Page 296
17 Microarrays and quantitative real-time reverse transcriptase–polymerase chain reaction......Page 298
Microarrays......Page 299
Correlation of microarray data from different platforms......Page 301
Microarray and qPCR: fold changes......Page 302
Unregulated genes......Page 304
Complex tissues – dilution effects......Page 307
Conclusions......Page 308
References......Page 309
In the beginning......Page 312
SNPs......Page 313
CCM, RFLP, and TaqMan......Page 314
Allelic-specific PCR......Page 315
GoldenGate and MIP......Page 316
References......Page 317
History of ancient DNA and PCR......Page 320
Sensitivity and the issue of contamination......Page 322
The advantages of PCR......Page 325
Practical applications of PCR in ancient DNA......Page 327
Conclusions......Page 330
References......Page 331
Index......Page 337