PCR 2: a practical approach
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Oxford [u.a.]
IRL Press
1995
|
| Schriftenreihe: | The practical approach series
150 |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XXV, 332 S. Ill., graph. Darst. |
| ISBN: | 0199634254 0199634246 |
Internformat
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| 245 | 1 | 0 | |a PCR 2 |b a practical approach |c ed. by M. J. McPherson ... |
| 246 | 1 | 3 | |a Polymerase chain reaction 2 |
| 264 | 1 | |a Oxford [u.a.] |b IRL Press |c 1995 | |
| 300 | |a XXV, 332 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 490 | 1 | |a The practical approach series |v 150 | |
| 650 | 4 | |a DNA replikacija | |
| 650 | 4 | |a Polimerozna verižna reakcija | |
| 650 | 4 | |a DNA replication | |
| 650 | 4 | |a Polymerase chain reaction | |
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Datensatz im Suchindex
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|---|---|
| adam_text | j CR2
A Practical Approach
Edited by
M J McPHERSON
Department of Biochemistry and Molecular Biology,
University of Leeds
B D HAMES
Department of Biochemistry and Molecular Biology,
University of Leeds
and
G R TAYLOR
Regional DNA Laboratory, Leeds
OXFORD UNIVERSITY PRESS
Oxford New York Tokyo
Contents
List of contributors
Abbreviations
1 Optimizing PCR 1
K K Kidd and G Ruano
1 Perspectives ; 1
The first few cycles 3
The exponential amplification phase 5
The plateau phase 5
The strategy for successful PCR 6
Contamination 7
2 Example of a standard protocol 7
3 Primer design 8
4 Optimization of reaction conditions 8
Primer sequence 8
Annealing temperature 9
Reaction buffer 9
Deoxynucleotide concentration 10
Thermostable DNA polymerase 10
Enzyme stabilizers 11
Concentration of primers 11
DNA template 11
Primer to template ratio 13
Number of target loci in a given template 13
Cycle profile for amplification 13
Addition of denaturation reagents to the reaction mixture IS
5 Detailed optimization protocol IS
6 Troubleshooting - ^ 18
No PCR product; an empty lane 19
Too many bands 20
Lots of primer-dimer 20
Product of the wrong size 21
PCR product with one template but not another 21
References 21
Contents
2 Use of speciality phosphoramidites in PCR 23
* MichaelJ McLean
1 Introduction 23
2 Reagents for the preparation of two oligomers per
synthesis (TOPS™) 24
The TOPS concept 24
The introduction of redundancies or modified residues at the
3 -end of oligomers 29
3 Phosphoramidites for the preparation of primers with
non-amplifiable tails 31
4 Reagents for the introduction of biotin 33
5 Psoralen reagents for use in DGGE and TGGE 35
References 37
3 Chemical methods for 5 non-isotopic
labelling of PCR probes and primers 39
Alex Andrus
1 Introduction 39
2 Synthesis, purification, and analysis of oligonucleotides 40
Automated oligonucleotide (primer/probe) synthesis 40
Cleavage and deprotection of oligonucleotides 42
Analysis, purification, and quantification 44
3 Fluorescent dye labelling 47
Fluorescent dye NHS coupling 47
Fluorescent dye phosphoramidite labelling 48
4 Enzyme labelling of oligonucleotides 49
5 Biotin-labelledoligonucleotides 50
6 Digoxigenin-labelled oligonucleotides 51
Acknowledgements 52
References 53
4 Solid phase PCR 55
Stefan Stamm and Jtirgen Brosius
1 Introduction 55
2 Coupling of oligonucleotides to a solid phase 56
Introduction 56
Contents
Choice of the solid phase 57
The coupling reaction 58
3 Synthesis of solid phase coupled cDNA 60
Reverse transcription of RNA using solid phase coupled
oligonucleotides 60
Monitoring of cDNA synthesis 61
Addition of homopolymers to the coupled oligonucleotides 62
4 Use of solid phase coupled cDNA in PCR experiments 63
5 Isolation and reverse transcription of RNA using solid
phase coupled primers 64
6 Inclusion of an oligonucleotide with an aminolink does
not interfere with the PCR reaction 67
7 Precautions and troubleshooting 67
8 Discussion 67
Acknowledgements 69
References 69
5 Solid phase sequencing of PCR products 71
Johan Wahlberg, Thomas Hultman, and Mathias Uhlen
1 Introduction 71
2 Different methods for DNA sequencing of PCR products 71
3 The principle of solid phase sequencing 75
4 Amplification of plasmid vector inserts for solid phase
sequencing 75
5 Amplification of genomic DNA for solid phase sequencing 79
Biotinylated primers 79
Amplification and isolation of single-stranded target DNA 81
6 Solid phase sequencing 81
7 Discussion 85
Acknowledgement 86
References 86
6 cDNA cloning by RT-PCR 89
Jean Baptiste Dumas Milne Edwards, Philippe Ravassard,
Christine Icard-Liepkalns and Jacques Mallet
1 Introduction 89
xi
Contents
=» 2 Reverse transcription 90
} General considerations 90
Isolation of RNAs 91
Quality control 92
cDNA synthesis • • 94
PCR on ss-cDNA 97
3 Anchored PCR 99
General considerations 99
Removal of the primer and RNA hydrolysis 100
Tailing-mediated anchored PCR 102
Ligation-mediated anchored PCR: SLIC strategy 103
Other uses of the SLIC strategy 107
Further improvement in cloning 5 -ends of mRNA 107
4 Cloning PCR products 108
General considerations 108
Analysis of PCR products by Flying Southern 109
5 Subtractive hybridization (general considerations) 110
6 Cloning cDNAs of multigene families 111
General considerations 111
The choice of degenerate primers 112
cDNA synthesis 113
PCR with degenerate primers 113
Analysis of recombinants 116
Isolation of full length cDNA clones 118
References 118
7 Quantification of DNA and RNA by PCR 119
Jie Kang, Jochen E Kiihn, Peter Schdfer,
Andreas Immelmann, and Karsten Henco
1 Introduction , 119
2 TGGE analysis 120
3 Quantification of DNA 121
Synthesis of standard DNA 121
Determination of plasmid copy number 122
Determination of DNA copy number in clinical samples 125
4 Quantification of RNA 128
Synthesis of standard RNA 128
Quantification of mRNA 130
5 Summary 131
Acknowledgements 133
References 133
xii
Contents
8 PCR MIMICs: competitive DNA fragments
* for use in quantitative PCR 135
Paul D Siebert and David E Kellogg
1 Introduction 135
2 Generation of PCR MIMICs 136
Determination of PCR MIMIC yield 139
Choice of heterologous DNA sequence 140
3 Validation of the PCR MIMIC strategy 141
Efficiency of amplification 141
Competitive PCR 141
Quantitative analysis of changes in IL-lp mRNA 144
4 Comments on cycle parameters and quantification of PCR
yields 147
Number of amplification cycles 147
Quantification of PCR product yields 147
5 Extension of the MIMIC strategy 148
References • 148
9 In vitro expression of proteins from PCR
products 149
Scott A Lesley
1 Introduction 149
2 Preparation of PCR products for translation 150
Promoters - 150
Translation initiation signals 151
Translation termination signals 152
Purification of PCR products 153
3 In vitro translation of PCR products 154
Coupled transcription —translation in E coli S30 extract 154
In vitro transcription of mRNA 155
Translation in rabbit reticulocyte Iysate 157
Translation in wheat germ Iysate 157
Assay of products 158
4 Applications of in vitro translation products 160
References 163
xiii
Contents
10 PCR-based approaches to human genome
mapping 165
JFJ Morrison and A F Markham
1 Introduction
2 Chromosome localization
3 Preliminary YAC analysis
Screening YAC libraries
Isolation of yeast clones
Pulse field gel electrophoresis (PFGE) of YACs
4 Vectorette libraries
Strategies of vectorette PCR
Construction of vectorette libraries from YAC DNA
Construction of vectorette libraries from other DNAs
Nested PCR
Using vectorette 2
Sequencing vectorette products
5 Exon identification
Previous approaches
Exon trapping
Acknowledgements
References
11 Fingerprinting of DNA and RNA using
arbitrarily primed PCR 197
John Welsh, Manuel Perucho, Miguel Peinado, David Ralph,
and Michael McClelland
1 Introduction 197
Applications of arbitrarily primed PCR 197
The arbitrarily primed PCR reaction 198
2 Genomic fingerprinting of mammalian DNA 199
Application to cancer research 199
Arbitrarily primed PCR 200
DNA sequencing by PCR-based cycle sequencing 207
Chromosomal localization of cloned sequences 208
3 RNA fingerprinting by arbitrarily primed PCR 210
RNA arbitrarily primed PCR (RAP-PCR) 211
An approximate kinetic model of RAP-PCR 212
How large must a RAP-PCR experiment be? 213
RNA purification 214
Choice of primers 214
Nested RAP-PCR 214
References 217
xiv
Contents
12 Mutational analysis: known mutations 219
a Clive R Newton
i
1 Introduction 219
2 Substrate DNAs 219
3 Amplification refractory mutation system (ARMS) 223
The ARMS reaction 223
Primer design 224
Specificity of ARMS primers 227
Plasmid cassette system for primer and/or PCR optimization 229
Recent advances: multiplex ARMS 233
Recent advances: fluorescent ARMS using four-colour technology 235
Recent advances: primers with non-amplifiable tails 239
4 Allele-specific oligonucleotide (ASO) analysis 242
Dot blots • 242
Reverse dot blots 243
5 Competitive oligonucleotide priming (COP) 246
6 Primer extension sequence test (PEST) 248
7 Mutation detection using Taq 5 —»3 exonuclease activity 249
8 Mutation detection by introduction of restriction sites 249
9 Single nucleotide primer extension (SNuPE) for mutation
detection 252
Acknowledgements 252
References 252
13 Mutational analysis: new mutations 255
K Michaelides, R Schwaab, MRA Lalloz, W Schmidt, and
EGD Tuddenham
1 Introduction 255
2 Genomic DNA amplification 255
3 Single strand conformation polymorphism (SSCP) 257
Standard SSCP 257
Consecutive SSCP 260
PCR-SSCP multiplexed analysis: application to the factor VIII
gene 265
4 Direct sequencing of biotinylated PCR products using
streptavidin-coated magnetic beads in a solid support
system 267
5 Chemical cleavage detection of mismatched base pairs 270
xv
Contents
) 6 Denaturing gradient gel electrophoresis (DGGE) 275
Preliminary theoretical work 276
Optimizing the conditions for DGGE 278
Practical procedures 281
Comments on the DGGE protocol 284
7 Choice of method: DGGE, SSCP, or chemical cleavage 285
DGGE 286
SSCP 286
Chemical cleavage 286
8 Discussion and conclusions 286
References 287
14 Linear amplification for the in vivo study
of ligand/DNA interactions 289
Hans Peter Saluz and Jean-Pierre Jost
1 Introduction 289
2 Chemical and enzymatic probes for genomic
footprinting 291
3 Genomic footprinting with dimethyl sulphate (DMS) 292
Preparation of cell suspensions 292
Preparation of nuclei 293
Treatment in vivo with DMS 294
Preparation of DNA 296
Restriction digestion of the genomic DNA 298
Cleavage reaction of the chemically modified genomic DNA
and sequencing reactions of the control DNA 298
4 Linear amplification 302
Selection of the oligonucleotide primer - 302
Labelling of the oligonucleotide primer 303
Determination of the melting temperature 304
Suitable enzymes for linear amplification 305
The linear amplification reaction 305
Purification and electrophoresis of the amplification products 306
References 307
15 Ligation-mediated PCR 309
Paul A Garrity, Barbara Wold, and Paul R Mueller
1 Introduction 309
2 Procedures for LMPCR 310
Preparation of linker and radiolabelled primer 3 310
xvi
Contents
First strand synthesis and Iigation of the linker 312
The LMPCR amplification reaction 312
3 Important criteria 316
Gene-specific primers 316
Unidirectional linker 317
Vent DNA polymerase 317
4 Expected results 317
5 Troubleshooting 320
High background 320
Short ladders 321
No ladder/weak ladder 321
Spurious bands 321
Fuzzy ladders/difficult to see footprints 321
Acknowledgements 322
References 322
Appendix 1 Suppliers of specialist items 323
Index 327
xvu
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| series | The practical approach series |
| series2 | The practical approach series |
| spelling | PCR 2 a practical approach ed. by M. J. McPherson ... Polymerase chain reaction 2 Oxford [u.a.] IRL Press 1995 XXV, 332 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series 150 DNA replikacija Polimerozna verižna reakcija DNA replication Polymerase chain reaction Polymerase-Kettenreaktion (DE-588)4256726-9 gnd rswk-swf Polymerase-Kettenreaktion (DE-588)4256726-9 s DE-604 MacPherson, Michael J. Sonstige oth The practical approach series 150 (DE-604)BV007921321 150 HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=006833537&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | PCR 2 a practical approach The practical approach series DNA replikacija Polimerozna verižna reakcija DNA replication Polymerase chain reaction Polymerase-Kettenreaktion (DE-588)4256726-9 gnd |
| subject_GND | (DE-588)4256726-9 |
| title | PCR 2 a practical approach |
| title_alt | Polymerase chain reaction 2 |
| title_auth | PCR 2 a practical approach |
| title_exact_search | PCR 2 a practical approach |
| title_full | PCR 2 a practical approach ed. by M. J. McPherson ... |
| title_fullStr | PCR 2 a practical approach ed. by M. J. McPherson ... |
| title_full_unstemmed | PCR 2 a practical approach ed. by M. J. McPherson ... |
| title_short | PCR 2 |
| title_sort | pcr 2 a practical approach |
| title_sub | a practical approach |
| topic | DNA replikacija Polimerozna verižna reakcija DNA replication Polymerase chain reaction Polymerase-Kettenreaktion (DE-588)4256726-9 gnd |
| topic_facet | DNA replikacija Polimerozna verižna reakcija DNA replication Polymerase chain reaction Polymerase-Kettenreaktion |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=006833537&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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