Molecular Diagnostics (eBook)

Part 1: Technical Backgrounds and Quality Aspects

W.B. van Leeuwen, E. van Pelt-Verkuil, R. te Witt (Herausgeber)

eBook Download: PDF

2019 | 1st ed. 2019
VII, 457 Seiten
Springer Singapore (Verlag)
978-981-13-1604-3 (ISBN)

Infos

The books Molecular Diagnostics Part 1 and 2 provide a comprehensive and practical overview of the state-of-the-art molecular biological diagnostic strategies that are being used in a wide variety of disciplines.

The editors and experts in their respective fields have combined their knowledge to write these two books. Many years of experience in the development, application and quality control of molecular diagnostic methods is reflected herewith.

Molecular Diagnostics Part 1 is dedicated to the theoretical backgrounds of the technologies often applied in molecular diagnostics, in which nucleic acid amplification methods (such as real-time PCR), sequencing and bioinformatics are the basic tools. The assay design and -development, combined with items of trouble-shooting are described in detail. As a foundation of reliable molecular diagnostic assays, the quality control required for validation, implementation and performance of molecular diagnostic assays is thoroughly discussed. This book also provides extensive information for those working with molecular techniques in a wide variety of research applications using conventional and real-time PCR technology, Sanger and high throughput sequencing techniques, and bioinformatics.

Molecular Diagnostics Part 2 highlights the applications of the molecular diagnostic methods in the various diagnostic laboratories, comprising:

- Clinical microbiology

- Clinical chemistry 

- Clinical genetics

- Clinical pathology 

- Molecular hematopathology

- Veterinary health

- Plant health

- Food safety

Both full-colour and well-illustrated books are particularly valuable for students, clinicians, scientists and other professionals who are interested in (designing) molecular diagnostic methods and for those who wish to broaden their knowledge on the current molecular biological revolution. The information in the books highlights the trend of the integration of multiple (clinical) disciplines into one universal molecular laboratory. 

E. van Pelt-Verkuil
Elizabeth van Pelt-Verkuil, born in Rotterdam, The Netherlands has been involved in the development of new techniques in both molecular - and cell biology for more than 40 years. From the first PCRs in 1985 up to now, Elizabeth participated in its development and the applicability in molecular diagnostics. First in pathology, later combined with microbiology. In the meantime her focus changed from research to diagnostics. After graduation in cell biology and biochemistry at University Leiden in 1975, Elizabeth got her PhD on (experimental) developmental biology in 1979, followed by a post-doc position on experimental arteriosclerosis. From 1982 onward, she was a lecturer and applied scientist at the University of Applied Science Leiden for both graduate and post-graduate education in i.e. molecular diagnostics. Elizabeth organised her first theoretical and practical course on PCR and in situ hybridisation for post-graduates in 1987.
Elizabeth was founder of the Centre of Bioscience and Diagnostics in 2001; a department of the before mentioned institute, specialised in post-graduate education. Elizabeth organised and participated in many courses and in-company-trainings on PCR and molecular diagnostics.
During a 5 -year period from 2011 onwards at the Professorship Innovative Molecular Diagnostics at the University of Applied Science Leiden, Elizabeth focused on the implementation and embedding of new laboratory developed qPCR tests for the detection and identification of viral pathogens, including all aspects of quality control. Since 2016 Elizabeth is owner of and consultant at 'Quality and Innovations in Molecular Diagnostics', a company which is specialised in implementation, quality control and education in the field of molecular diagnostics.
Elizabeth van Pelt-Verkuil is a member of the Dutch Society of Medical Microbiology (NVMM) and participates in the Study group of Molecular Diagnostics of Infectious Diseases (WMDI).

Elizabeth van Pelt-Verkuil is editor/author of 2 Dutch books on Molecular Diagnostics and a textbook on PCR.

E. van Pelt-Verkuil et.al. Moleculaire Diagnostiek (editors/author) (1e Ed; in Dutch) 2001. Bohn Stavleu van Loghum; ISBN9-789031331031
E. van Pelt-Verkuil et.al. (author) Principles and technical aspects of PCR amplification (2008). Springer ISBN 9 781402062407
E. van Pelt-Verkuil and W.B. van Leeuwen, Moleculaire Diagnostiek (editors/author) (2nd Ed; in Dutch) 2013. Syntax media; ISBN 9789077423950

W.B. van Leeuwen
Willem van Leeuwen was born  in Rotterdam, The Netherlands. He studied Microbiology on the University of Applied Science in Rotterdam (BSc in 1975) and specialized in Medical Biology on the University of Applied Science in Etten-Leur, The Netherlands (MSc in 1982). In 2002 he wrote his thesis 'Binary typing of Staphylococcus aureus' and became a specialist in Medical Molecular Microbiology at the Erasmus Medical Center Rotterdam, department of Medical Microbiology & Infectious Diseases. The population structure analysis and host-pathogen interaction of Staphylococcus aureus were the subjects of study during the post-doctoral phase in this department. From august 2002 through 2007 he is responsible for the unit Molecular Diagnostics (validation and implementation of new diagnostic platforms). In February 2007 he was invited to become professor Innovative Molecular Diagnostics at the University of Applied Science of Leiden, the Netherlands and became manager of the research center, 'Toplab' in Leiden. In September 2013, he was appointed as professor at the University of Applied Science Leiden. Since september 2017 he became senior professor of the Leiden Centre for Applied Bioscience at the University of Applied Science Leiden.

Since 1996, Willem is a member of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Dutch Society of Medical Microbiology (NVMM) and the Study group of Molecular Diagnostics of Infectious Diseases (WMDI).
Willem is an international scientific Expert for diagnostics and subtyping of MRSA for QCMD (Quality Control Molecular Diagnostics) since 2005.
Since 2011, Willem is staff member / instructor of the Dutch National Committee for Medical-Biological Research Training (MMO) in the Netherlands.
Last, but not least, Willem is honorary member of the Iranian Society of Microbiologists since 2010.

Willem is editor/author of multiple books in the field of Molecular Diagnostics:

1. E. van Pelt-Verkuil and W.B. van Leeuwen (editors/author). Moleculaire Diagnostiek. (2nd Ed; in Dutch) 2003. Syntax media; ISBN 9789077423950
2. W.B. van Leeuwen and C. Vink (editors/author). Molecular Diagnostics: techniques and applications. IVA publishers, 2009.
3. John P. Hays and W.B. van Leeuwen Eds. The Role of New Technologies in Medical Microbiological Diagnosis and Research. 2012. Bentham Books publishers. elBN:978-1-60805-316-2.

R. te Witt
Ren? te Witt is born in Rotterdam, The Netherlands and is a real 'Rotterdammert'. He studied Medical Microbiology and worked as a technician in a diagnostic laboratory for several years, all in Rotterdam. In 2007 he transferred to the Erasmus MC, also in Rotterdam, to write his thesis 'Clinical Microbiological Diagnostics 2.0'. After finishing his PhD in 2012, he took charge of the subunit Molecular Diagnostics at the department of Medical Microbiology and Infectious Diseases of the Erasmus MC. Here, he was responsible not only for the day-to-day routine diagnostics, but also for development and implementation of new molecular assays, quality assurance and research projects,. 
During 2012, Ren? started his training to become Medical Molecular Microbiologist (MMM), both at the Erasmus MC and the LUMC in Leiden. In 2015 he graduated as MMM and moved to Rijswijk to work as MMM at NMDL (Netherlands Molecular Diagnostics Laboratory), a service laboratory specialized in molecular diagnostics in medical microbiology, virology and pathology.

Ren? is a member of the Dutch Society of Medical Microbiology (NVMM) since 2007 and is one of the editors of the Netherlands Magazine for Medical Microbiology (NTMM) since 2013.
Furthermore, Ren? is member of and participates in the Study group of Molecular Diagnostics of Infectious Diseases (WMDI) since 2007.

Ren? joined the editorial team of this book in 2015 because of his excellent skills and broad experience in modern molecular diagnostics in combination with his current position in a rapid expanding laboratory using state-of-the-art molecular diagnostic techniques.

Inhalt

Preface 5
References 6
Contents 7
1 Introduction, The Importance of Nucleic Acids in Diagnostics 8
Abstract 8
1.1 Introduction 8
1.2 Nucleic Acids and Molecular Diagnostics 9
1.3 Layout of This Book 11
1.4 Future 13
Reference 13
2 Background and Analysis of Nucleic Acids 14
Abstract 14
2.1 Introduction 14
2.2 Eukaryotic DNA 16
2.2.1 Coding Versus Non-coding Sequences 16
2.2.2 Single-Nucleotide Polymorphism (SNP) 19
2.2.3 ‘CpG Sites’ 20
2.2.4 Genetic, Congenital and Acquired Disease-Causing Aberrations in the Genome 20
2.3 Nucleic Acids of Bacteria and Archaea 21
2.4 Nucleic Acids of Viruses 22
2.5 RNA of Pro- and Eukaryotes 24
2.5.1 Messenger RNA (mRNA) 26
2.5.2 Transfer RNA (tRNA) 29
2.5.3 Ribosomal RNA (rRNA) 29
2.5.4 Non-coding RNAs 30
2.5.5 RNA and the Polymerase Chain Reaction (cDNA) 31
2.6 Isolation and Purification of Nucleic Acid and Quality Control of Isolated DNA/RNA 32
2.6.1 Nucleic Acid Isolation and Purification 33
2.6.1.1 DNA Isolation and Purification 33
2.6.1.2 RNA Isolation and Purification 36
2.6.1.3 The Boom Method 37
2.6.2 PCR Interfering Factors 37
2.6.2.1 Inhibition of Polymerase Activity During Amplification 38
2.6.2.2 Integrity of Nucleic Acids in Paraffin-Embedded Samples 39
2.6.3 Quality Criteria for DNA and RNA Isolates 41
2.6.4 Electrophoresis 41
2.6.5 UV Spectrometry 43
2.7 Detection of Nucleic Acids 43
2.7.1 Concentration Determinations with Fluorescent Dyes 43
2.7.1.1 Detection of Nucleic Acids 43
2.7.1.2 Concentration Determination of Nucleic Acid with Fluorescent Dyes 44
2.7.1.3 Discrimination of Nucleic Acids Based on Binding of Dyes 45
2.7.2 Determination of Nucleic Acids with Fluorescent DNA and RNA Dyes 46
2.8 Techniques to Analyse Nucleic Acids 49
2.8.1 Electrophoresis Techniques 49
2.8.1.1 Electrophoresis Matrix 49
2.8.1.2 Special Applications of Electrophoresis 51
2.8.2 Hybridisation (Chaps. 4 and 9) 53
2.8.3 Amplification of Nucleic Acids (Chaps. 5 and 7) 54
2.8.4 DNA Sequence Analysis (Chap. 8) 56
2.8.5 Biophysical Methods (Chap. 10) 56
References 56
3 Primers and Probes 58
Abstract 58
3.1 Introduction 59
3.1.1 Probe Hybridisation 59
3.1.2 Primer Hybridisation (Annealing) 60
3.2 Primer and Probe Design 60
3.2.1 Primer Design 61
3.2.2 Primer Design Guidelines 63
3.2.3 Primer and Probe Design 65
3.2.4 Challenges in Primer/Probe Design 66
3.2.4.1 Mismatch Tolerance 66
3.2.4.2 Mismatch Discrimination 68
3.2.5 Probe Design Guidelines 69
3.3 Preparation of Primers and Probes 70
3.3.1 Chemical Synthesis of Oligonucleotides (Oligos) 70
3.3.2 Enzymatic Synthesis of Probes via PCR Amplification 72
3.3.3 Cloning Using of Recombinant DNA Techniques 73
3.4 Labels, Incorporation and Detection of Labelled Nucleotides in Primers and Probes 73
3.4.1 Labels: Recognition and Detection of Hybridisation and PCR Products 73
3.4.2 Incorporation of Labels 74
3.4.2.1 Chemical Synthesis 75
3.4.2.2 Chemical Labelling 76
3.4.2.3 Enzymatic Labelling Methods 76
3.4.2.4 Nick Translation 78
3.4.2.5 ‘Random’ Primer Labelling 78
3.4.2.6 End Labelling 79
3.4.3 Detection of Labels 80
3.4.4 Haptens 81
3.4.5 Reporters 83
3.5 Different Types of Primers 85
3.5.1 Extended Primers 85
3.5.2 Degenerated/Consensus Primers 88
3.5.3 Translocation Primers 90
3.5.4 Multiplex PCR 90
3.5.5 Repeat Primers 91
3.6 Probes in Molecular Diagnostics 91
3.6.1 Probes for Real-Time PCR 92
3.6.1.1 Hydrolysis Probe (‘Quenching’) 92
3.6.1.2 Molecular Beacon 92
3.6.1.3 ‘Dual’ Probe (Enhancement) 96
3.6.2 Nucleic Acid Derivatives and -Analogues in Primers and Probes 96
3.6.2.1 Peptide Nucleic Acid (PNA) 96
3.6.2.2 Locked Nucleic Acid (LNA) 97
3.7 Specific and Non-specific Chemistry in Real-Time PCR: Intercalation or Labelled Primers and Probes 97
3.7.1 Non-specific Chemistry 98
3.7.2 Specific Chemistry 99
References 102
4 Basic Principles of Nucleic Acid Hybridisation 103
Abstract 103
4.1 Introduction 103
4.2 General Aspects of Classical Nucleic Acid Hybridisation 104
4.3 Denaturation and Renaturation of DNA and RNA 106
4.3.1 Denaturation of DNA and RNA 106
4.3.2 Renaturation of DNA and RNA 108
4.4 Hybridisation of Nucleic Acids Using DNA or RNA Probes 109
4.5 Specificity of the Probe-Target Binding Stringency and Tm
4.6 Stringency of Probe-Target Binding During Washing Steps for Probes? greaterthan ?100 Bases 114
4.6.1 Washing 114
4.7 Hybridisation of Primers and Oligoprobes the Use of the Tm
4.7.1 Characteristics of Oligonucleotide (Oligo) Hybridisation 115
4.7.2 Primer Hybridisation or Annealing 117
4.7.3 Multiplex PCR 118
4.7.4 Design for Hybridisation/Primer Annealing 119
4.7.5 Probes for Real-Time PCR 120
4.7.6 The Use of the Tm to Characterise Amplicons 121
4.8 Practical Implementation of Conventional Hybridisation 121
4.8.1 Pre-treatment 122
4.8.2 Blocking and Prehybridisation 122
4.8.3 Hybridisation Mixture 122
4.8.4 Hybridisation Time 123
4.8.5 Final Steps After Hybridisation 123
4.9 Hybridisation Methods 124
4.9.1 Classical Hybridisation in Solution 124
4.9.2 Hybridisation to a Solid Phase 124
4.9.3 In Situ Hybridisation 129
4.9.3.1 Practical Problems for In Situ Hybridisation 129
4.9.3.2 In situ Hybridisation: Fixation and Preservation of Nucleic Acid Targets 131
4.10 Bead-Based Flow Cytometry for the Medical Microbiological Diagnostics 134
4.11 Summary 134
References 135
5 Principles of PCR 136
Abstract 136
5.1 Introduction 137
5.2 The Principle of PCR 138
5.3 Implementation of PCR General Principles
5.4 The PCR Cycle 143
5.4.1 Denaturation 145
5.4.2 Primer-Annealing Phase and Annealing Temperature (Ta) 146
5.4.3 Elongation of Primers or Extension Phase 146
5.5 Amplimer Synthesis During PCR 147
5.5.1 Different Phases of DNA Synthesis During PCR 148
5.5.1.1 Phase 1: Ground Phase 149
5.5.1.2 Phase 2: Exponential Phase 151
5.5.1.3 Phase 3: Transition or Interphase 155
5.5.1.4 Phase 4: Plateau Phase 156
5.5.1.5 Melting Curve Analysis 158
5.5.2 The Calibration Curve and the Standard Curve 159
5.5.2.1 The (Real-Time) qPCR-Calibration Curve 159
5.5.2.2 The Standard Curve and qPCR Efficiency 160
5.6 Conduct of PCR (Conventional and QPCR) 163
5.7 Real-Time PCR (QPCR) 164
5.8 Sensitivity of the PCR 166
5.9 The Individual Elements of the Reaction Mixture for PCR 168
5.9.1 DNA: Extraction and Origin 168
5.9.2 Primers 168
5.9.3 The Nucleotide Triphosphates 168
5.9.4 PCR Buffer, Monovalent and Divalent Ions for Taq-DNA Polymerase 171
5.9.5 Internal Amplification and Process Controls 171
5.9.6 Additives 173
5.9.7 Fluorochromes and Probes 174
5.9.8 Taq-DNA Polymerase, Variants and Other Heat-Stable Enzymes 175
5.10 Optimisation, Reaction Specificity and the Amplification Program 181
5.11 Analysis of PCR Products with Conventional PCR 184
5.11.1 Size Determination of Amplimers Using DNA Dyes and Electrophoresis 184
5.11.2 Hybridisation of PCR Products with Labelled Probes 184
5.11.3 DNA Sequencing 186
5.12 Analysis and Application of Specialised PCR Techniques Using Conventional and/or Real-Time PCR 191
5.12.1 ‘Time-Release’ (‘Hot Start’) (Q)PCR 191
5.12.2 ‘Touchdown’- and ‘Touch-Up’-PCR 193
5.12.3 Multiplex PCR 194
5.12.4 Inverse PCR 195
5.12.5 Analysis of Differences in Bases Using ASO and ARMS Techniques 196
5.12.6 Repeat and Inter-repeat PCR and Amplification Fragment Length Polymorphism (AFLP) 197
5.12.6.1 Repeat PCR 197
5.12.6.2 Inter-Repeat PCR 198
5.12.7 Nested PCR 202
5.12.8 Multiplex Ligation-Dependent Probe Amplification (MLPA®) 202
5.12.9 Methylation-Specific PCR 204
5.12.10 Bridge Amplification for Sequencing 208
5.13 Analysis of PCR Products with Real-Time PCR 208
5.13.1 Absolute Quantification (QPCR) 208
5.13.2 Relative Quantification (RT-QPCR) 211
5.13.3 The Standard Curve and Melting Curve Analysis as Optimisation Control 213
5.13.4 High-Throughput Amplification Techniques 214
5.13.5 Digital PCR (dPCR) 214
5.13.6 High-Resolution Melting Curve Analysis (HRMA) 215
References 218
6 Quality Assurance, Management and Control in Molecular Diagnostics 221
Abstract 221
6.1 Introduction: Validation Algorithm 222
6.2 Phase 1: Quality Control in PCR Technology: Organisation and Management 227
6.2.1 Organisation and Validation of a Molecular Diagnostic Laboratory 228
6.2.2 Certification (Validation) of Key Reagents 229
6.2.3 Preventing and Identifying Contamination 231
6.3 Phase 2: The Second Level of Quality Control in PCR Techniques: The Design of (Multiplex) PCR, Technical Validation and Implementing 238
6.3.1 Extrinsic and Intrinsic Factors and Optimisation of a Specific Target 238
6.3.2 Development of In-House Assays/Laboratory Developed Tests (Phase 2A) 239
6.3.2.1 PCR Conditions: First PCR Optimisation Experiments and Troubleshooting 245
6.3.2.2 Positive and Negative Controls in Step 2A 256
6.3.2.3 Amplification Control 259
6.3.2.4 Standards and References 259
6.3.2.5 Issues with RNA Targets 262
6.3.3 Technical Validation with Clinical Samples: Controls and Use of Retrospective Samples (Step 2B/C) 264
6.3.3.1 Amplification of Process Controls 264
6.3.3.2 Molecular Markers in Retrospective and Prospective Investigations 268
6.3.3.3 Complex Genomic Context and the Influence of the Sample Matrix and Environmental Compounds on PCR Results 268
6.3.3.4 Efficiency of PCR 269
6.3.3.5 Sample Transport 270
6.3.3.6 Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) 270
6.3.3.7 Other Indications to Phase 2 271
6.3.4 Quality Criteria for Technical Validation 274
6.3.4.1 Technical Validation of In-House Assays 276
6.3.4.2 Technical Specificity and Sensitivity 277
6.3.4.3 AMR (Analytical Measurement Range), CRR (Clinically Reportable Range) and RR (Reportable Range) Theoretically
6.3.4.4 Determination of AMR (Dynamic/Analytical Range) in Practice 280
6.3.4.5 Determination of the Reportable Range in Practice (RR) 281
6.3.4.6 Efficiency 284
6.3.4.7 Accuracy (Trueness) 284
6.3.4.8 Systematic Errors 287
6.3.4.9 Random Errors 287
6.3.4.10 Robustness 288
6.3.4.11 Reproducibility 289
6.3.4.12 Repeatability 290
6.3.4.13 Recovery 290
6.3.4.14 First Level of Concordance 290
6.3.4.15 Decision for the Validation of an Assay in Prospective Studies 292
6.3.5 Clinical (Diagnostic) Validation 292
6.3.6 Diagnostic Sensitivity and Specificity, Positive and Negative Predictive Values 294
6.3.6.1 The Concrete “Yes or no Answer” Two Separate Populations
6.3.6.2 No Absolute Answer Possible Partly Overlapping Populations
6.3.6.3 The ROC Curve (Receiver Operator Characteristic) 299
6.3.6.4 The Predictive Value of a Diagnostic Test 302
6.3.6.5 Second Level of Concordance 305
6.3.6.6 Verification 306
6.3.6.7 Summary of Phases 1 and 2 307
6.4 The Third Level of Quality Control in PCR Techniques: Maintaining Quality During Routine Application of a Molecular Diagnostic Assay 309
6.4.1 Quality Assurance and the Human Factor 310
6.4.2 First-Line Control 312
6.4.3 Westgard Rules and Levey–Jennings Control Chart 313
6.4.4 Second-Line Control 316
6.4.5 Third-Line Control Quality Assessment of Laboratories by Proficiency Studies (EQA
6.4.5.1 Design and Planning 319
6.4.5.2 Quality Control of the Test Panels 320
6.4.5.3 Distribution of Proficiency Panels Testing and Reporting by Participating Laboratories
6.4.5.4 Data Analysis and Publication of Results 320
6.4.5.5 PDCA Cycle for Participating Laboratories 320
6.4.6 Accreditation and Meeting of Quality Standards 321
References 322
7 Quantitative Isothermal Molecular Amplification Techniques 324
Abstract 324
7.1 Introduction 324
7.2 Signal Amplification Techniques 325
7.2.1 Branched DNA Technology 325
7.2.2 Hybrid Capture Technique 327
7.3 Isothermal Target Amplification Techniques 328
7.3.1 Nucleic Acid Sequence-Based Amplification (NASBA) 328
7.3.2 Transcription-Mediated Amplification (TMA) Using Reverse Transcriptase 330
7.3.3 Strand Displacement Amplification (SDA) 332
7.3.4 LAMP Technology 337
References 340
8 DNA Sequencing 341
Abstract 341
8.1 Introduction 341
8.2 Sanger Sequencing 342
8.2.1 The Chain Termination Principle 342
8.2.2 Improvements to Sanger Sequencing 344
8.3 Next-Generation Sequencing 344
8.3.1 Flow Cell Technology 345
8.3.2 Sequencing Chemistry 346
8.3.3 Signal Detection 347
8.3.4 Illumina Platforms 348
8.3.4.1 Additional Illumina Features 349
8.4 Third-Generation Sequencing (Single-Molecule Sequencing) 350
8.4.1 PacBio Sequencing 351
8.4.2 Nanopore Sequencing 354
8.5 Applications 356
8.5.1 Clinical Microbiology (Bacteriology) 356
8.5.2 Targeted NGS (Amplicon Sequencing) 357
8.5.3 Clinical Microbiology (Virology) 358
8.5.4 Transcriptome Sequencing 358
8.5.5 Clinical Genetics 359
8.6 Conclusion 360
8.7 The Future of DNA Sequencing 361
Recommended Literature 361
Online Sources 362
9 Bioinformatics 363
Abstract 363
9.1 Introduction 364
9.2 NGS Data Generation and Processing 364
9.2.1 Selecting and Testing Pipeline Components 367
9.2.2 Re-sequencing and Array Experiments in Diagnostics 367
9.3 Reference Sequences 367
9.4 Sequence Annotation 368
9.5 Detection of Sequence Variants 369
9.6 Primer Design in Hereditary Disease Diagnostics 370
9.7 Searching Sequences in Databases 370
9.8 Help Reporting Sequence Variants 371
9.9 Information About Genetic Conditions 371
9.10 Information About Gene Variants 373
9.11 Potential Effects of Sequence Variants 373
9.12 Analysis and Prediction of Variants 374
9.13 Prediction of Alterations in Splicing 374
9.14 Prediction of Altered Protein Sequences 374
9.15 Effects of Amino Acid Changes 375
9.16 The Importance of Correct Predictions 375
9.17 Structural Variants 376
9.18 Interpretation of Diagnostic Microarray Data 376
9.19 Databases with Structural Genome Variation 377
9.20 Work Flows and Web Services 377
9.21 Application of the Programs Described in Diagnostics 378
9.22 Hints and Warnings 379
Appendix 1 379
Appendix 2 382
Recommended Literature 388
10 The Ongoing Revolution of MALDI-TOF Mass Spectrometry for Molecular Diagnostics 389
Abstract 389
10.1 Introduction 390
10.2 Basics TOF Mass Spectrometry 390
10.2.1 Brief History 390
10.2.2 MALDI-TOF MS Basics 392
10.2.3 The Ion Formation Mechanism 392
10.2.4 Consequences for Spotting the Sample 394
10.2.5 MALDI Matrices 395
10.2.6 Protocols and Spot Preparation 395
10.2.7 TOF Mass Spectrometer Principles 397
10.2.7.1 Basic Principle 397
10.2.7.2 Fine-Tuning, Ion Optics and Delayed Extraction 399
10.3 Ion Detection and Signal Processing 401
10.3.1 Electron Multiplier Tube 401
10.3.2 Micro-channel Plate Detector 401
10.3.3 Signal Processing 403
10.3.4 Classification of Spectra Using MALDI-TOF MS 405
10.3.5 Reference Database 408
10.4 Future Directions for MALDI-TOF 408
10.4.1 Further Automation 409
10.4.2 Database Development 409
10.4.3 Current Limitations, New Functionality 409
References 410
11 Information in the Amplification Curve 412
Abstract 412
11.1 Introduction 412
11.2 MIQE Guidelines 414
11.3 The Principle of Analysis of qPCR Data 415
11.3.1 Kinetics of PCR 415
11.3.2 The Individual Amplification Curve 416
11.3.3 Data Analysis Principle 418
11.4 Estimation of the Analysis Parameters 418
11.4.1 Baseline Fluorescence 418
11.4.1.1 Trend Line 419
11.4.1.2 Fit of a Sigmoidal Curve (S-Curve) 419
11.4.1.3 Reconstruction of the Exponential Phase 421
11.4.2 Quantification Threshold (Fq) and Quantification Cycle (Cq) 421
11.4.2.1 ‘ThresholdCycle’ Method 422
11.4.2.2 Second Derivative Maximum Method 424
11.4.2.3 Mid-Point Method 424
11.4.2.4 Cy0 Method 425
11.4.3 PCR Efficiency 425
11.4.3.1 PCR Efficiency Derived from a Standard Curve 427
11.4.3.2 PCR Efficiency Derived from the Amplification Curves 430
11.4.4 Calculation of the Starting Concentration (N0) 432
11.4.4.1 S-Curve Based Approaches 433
11.4.4.2 LinRegPCR 433
11.4.4.3 Using the Mean PCR Efficiency 433
11.4.5 Quantification Without Determining the PCR Efficiency 434
11.4.6 Summary of QPCR Data Analysis 434
11.5 Assay Validation 435
11.5.1 Quality Controls Required to Validate Correct Amplification 435
11.5.1.1 Melting Curve Analysis 435
11.5.1.2 Gel Electrophoresis 436
11.5.1.3 Sequencing 436
11.5.1.4 Reaction Controls 436
11.5.2 The Individual Amplification Curves 437
11.5.2.1 Baseline Fluorescence 437
11.5.2.2 Plateau Phase 438
11.5.2.3 Exponential Phase 438
11.5.3 The Standard Curve 439
11.5.3.1 Data Points 439
11.5.3.2 Validation of the Standard Curve 439
11.5.3.3 Run-to-Run Variation 440
References 440
Glossary to Parts 1 and 2 442

Produkt-Details

• Erscheint lt. Verlag: 3.6.2019
• Zusatzinfo: VII, 457 p. 224 illus., 205 illus. in color.
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie ► Allgemeines / Lexika
• Themenwelt: Medizin / Pharmazie ► Medizinische Fachgebiete ► Laboratoriumsmedizin
• Themenwelt: Studium ► 2. Studienabschnitt (Klinik) ► Pathologie
• Themenwelt: Technik
• Themenwelt: Weitere Fachgebiete ► Land- / Forstwirtschaft / Fischerei
• Schlagworte: Amplification Techniques • Disease Characterization • Next Gen Sequencing • quality control • real-time PCR
• ISBN-10: 981-13-1604-X / 981131604X
• ISBN-13: 978-981-13-1604-3 / 9789811316043