Definitions and Scope of the Subject 25
Table of Contents
0. Executive Summary 23
1. Introduction 25
Definitions and scope of the subject 25
Historical evolution of molecular diagnostics 25
Molecular biology relevant to molecular diagnostics 26
DNA 26
DNA polymerases 26
Restriction endonucleases 27
DNA methylation 27
RNA 28
RNA polymerases 28
Non-coding RNAs 28
DNA transcription 29
Chromosomes 29
Telomeres 30
Mitochondrial DNA 30
Genes 31
The genetic code 31
Gene expression 31
The human genome 32
Variations in the human genome 32
Variations in DNA sequences 32
Single nucleotide polymorphisms 33
Haplotyping 33
Copy number variations in the human genome 34
Genotype and haplotypes 35
Insertions and deletions in the human genome 35
Complex chromosomal rearrangements 36
Large scale variation in human genome 36
Structural variations in the human genome 37
Replication of the DNA helix 37
Transposons 38
Proteins 38
Proteomics 38
Monoclonal antibodies 39
Aptamers 39
Basics of molecular diagnostics 39
Tracking DNA: the Southern blot 39
Pulsed-field gel electrophoresis 40
DNA Probes 40
The polymerase chain reaction 41
Basic Principles of PCR 41
Target selection 41
Detection of amplified DNA 41
Impact of human genome project on molecular diagnostics 42
Mapping and sequencing of structural variation from human genomes 42
1000 Genomes Project 43
Human Variome Project 44
Role of bioinformatics in molecular diagnostics 44
Systems biology approach to molecular diagnostics 45
Biomarkers 45
Applications of molecular diagnostics 45
2. Molecular Diagnostic Technologies 49
Introduction 49
DNA sample collection and extraction 49
Blood samples 49
Buccal swabs and saliva 49
Urine samples for transrenal DNA 50
Manual vs automated DNA extraction 50
Sample preparation 51
Pressure Cycling Technology 51
Membrane immobilization of nucleic acids 51
Automation of sample preparation in molecular diagnostics 52
ABI PRISM 6700 Automated Nucleic Acid Workstation 52
BioRobot technology 52
COBAS AmpliPrep System 52
GENESIS FE500 Workcell 52
GeneMole 53
PCR BioCube 53
QIAsymphony 53
Tigris instrument system 53
Techniques for sample preparation that are suitable for automation 54
Xtra Amp Genomic DNA Extraction 54
Extraction of DNA from paraffin sections 54
Dynabead technology 54
Pressure Cycling Technology 54
SamPrep 55
Use of magnetic particles for automation in genome analysis 55
Companies involved in nucleic acid isolation 56
Novel PCR methods 57
Addressing limitations of PCR 57
CAST-PCR 57
Combined PCR-ELISA 57
Convection PCR 58
Digital PCR 58
Emulsion PCR 59
ExCyto PCR 59
Long and accurate PCR 60
Multiplex PCR 60
Real-time PCR systems 60
Dyes used in real-time PCR 61
Commercially available real-time PCR systems 61
LightCycler PCR system 62
LightUp probes based on real-time PCR 62
READ™ real-time PCR method 63
StellARray™ technology 63
7500 Fast Dx Real-Time PCR Instrument 63
Applications of real-time PCR 63
Limitations of real-time PCR 64
Quantitative PCR for accurate low level DNA analysis 64
Guidelines for real-time qPCR 65
Future applications of real-time qPCR 65
Reverse transcriptase (RT)-PCR 66
Standardized reverse transcriptase PCR 66
Single cell PCR 67
LATE-PCR 67
COLD-PCR 67
AmpliGrid-System 68
Post-PCR genotyping methods 68
High-resolution melt analysis 69
Monitoring of gene amplification in molecular diagnostics 69
Non-PCR nucleic acid amplification methods 69
DNA probes with conjugated minor groove binder 69
Dynamic Flux Amplification 70
Isothermal reaction for amplification of oligonucleotides 70
ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids) 70
Linked Linear Amplification 71
Multiplex Ligation-Dependent Probe Amplification 71
Rapid analysis of gene expression 71
Rolling circle amplification technology 72
Gene-based diagnostics through RCAT 73
RCAT-immunodiagnostics 73
RCAT-pharmacogenomics 74
Circle-to-circle amplification 74
Ramification amplification method 74
Single Primer Isothermal Amplification 75
Transcription mediated amplification 75
WAVE nucleic acid fragment analysis system 75
Technologies for signal amplification 76
3 DNA dendrimer signal amplification 76
Hybridization signal amplification method 77
Signal mediated amplification of RNA technology 78
Invader assays 79
Hybrid Capture technology 80
Branched DNA test 81
Tyramide signal amplification 81
Non-enzymatic signal amplification technologies 81
Direct molecular analysis without amplification 82
Trilogy™ Platform 82
Direct detection of dsDNA 83
Multiplex assays 84
Fluorescent in situ hybridization 84
FISH technique 85
Applications of FISH 85
Modifications of FISH 86
Direct visual in situ hybridization 86
Direct labeled Satellite FISH probes 86
Comparative genomic hybridization 87
Primed in situ labeling 87
Interphase FISH 87
FISH with telomere-specific probes 88
Multicolor FISH 88
Simultaneous Ultrasensitive Subpopulation staining/Hybridization In situ 88
Automation of FISH 89
Companies involved in FISH diagnostics 89
RNA diagnostics 90
Branched-chain DNA assay for measurement of RNA 91
Cycling probe technology 91
Invader RNA assays 91
Linear RNA amplification 92
Non-isotopic RNase cleavage assay 92
Nucleic acid sequence-based amplification 92
Q Beta replicase system 94
RNAScope 94
Solid Phase Transcription Chain Reaction 95
Transcriptome analysis 95
Visualization of mRNA expression in vivo 95
MicroRNA diagnostics 96
Real-time PCR for expression profiling of miRNAs 96
Microarray vs quantitative PCR for measuring miRNAs 96
Use of LNA to explore miRNA 97
Nuclease Protection Assay to measure miRNA expression 97
Microarrays for analysis of miRNA gene expression 97
Modification of in situ hybridization for detection of miRNAs 98
Whole genome amplification 99
Companies that provide technologies for whole genome amplification 99
QIAGEN’s Repli-G system 99
GenomePlexÔ Whole Genome Amplification 100
DNA sequencing 100
Companies involved in sequencing 101
Applications of next generation sequencing in molecular diagnostics 103
Companies developing sequencing for molecular diagnostics 103
Genome-wide approach for chromatin mapping 104
Mitochondrial sequencing 105
Identification of unknown DNA sequences 105
Optical mapping 105
Gene expression analysis 106
Gene expression profiling on whole blood samples 107
Gene expression patterns of white blood cells 107
Gene expression profiling based on alternative RNA splicing 107
MAUI (MicroArray User Interface) hybridization 108
Monitoring in vivo gene expression by molecular imaging 108
Serial analysis of gene expression (SAGE) 108
Single-cell gene expression analysis 109
T cell receptor expression analysis 110
Tangerine™ expression profiling 110
Whole genome expression array 110
Ziplex™ system 111
Companies involved in gene expression analysis 111
Peptide nucleic acid technology 112
Use of PNA with fluorescence in situ hybridization 113
PNA and PCR 113
Use of PNA with biosensors 114
PNA-based PD-loop technology 114
PNA-DNA hybrid quadruplexes 115
Companies involved in PNA diagnostics 115
Locked nucleic acids 115
Zip Nucleic Acids 116
Electrochemical detection of DNA 117
Mediated nucleic acid oxidation 117
Detection of hybridized nucleic acid with cyclic voltametry 118
Electrochemical detection based on Toshiba’s CMOS technology 118
Concluding remarks on electrochemical DNA detection 118
Bead-based assay platforms 118
Scorpions™ technology 119
The Scorpions reaction 119
Applications of Scorpions 120
Nucleic acid lateral flow molecular diagnostics 120
3. Biochips, Biosensors, and Molecular Labels 123
Introduction to biochip technology 123
Applications of biochips in diagnostics 123
GeneChip 124
GeneChip Human Genome Arrays 125
AmpliChip CYP450 125
Electronic detection of nucleic acids on microarrays 125
Microchip capillary electrophoresis 126
Strand displacement amplification on a biochip 126
Rolling circle amplification on microarrays 126
LiquiChip-RCAT 126
Fast PCR biochip 127
Multiplex microarray-enhanced PCR for DNA analysis 127
Multiplexed Molecular Profiling 127
Universal DNA microarray combining PCR and ligase detection reaction 128
Genomewide association scans 128
Whole genome chips/microarrays 129
Transposon insertion site profiling chip 129
Standardizing the microarrays 130
Companies involved in developing biochip technology for diagnostics 130
Future of biochip technology for molecular diagnostics 131
Microfluidic chips 132
Fish-on-chip 132
Lab-on-a-chip 132
LabCD 133
Micronics' microfluidic technology 133
Microfluidic chips/arrays using PCR 133
Microfluidic automated DNA analysis using PCR 133
Digital PCR Array 133
Digital PCR on a SlipChip 134
Microfluidic chips integrated with RCAT 134
Microfluidic chips integrated with PET 134
Companies developing microfluidic technologies 135
Biosensor technologies 135
Classification of biosensor technologies 136
DNA-based biosensors 137
DNA hybridization biosensor chips 137
PCR-free DNA biosensor 137
DNA based biosensor to detects metallic ions 138
Genetically engineered B lymphocytes 138
Biosensors immunoassays 138
PNA (peptide nucleic acid)-based biosensors 139
Protein-based biosensors 139
Antibody biosensors 139
Cell-based biosensors (cytosensors) 139
Multicell biosensors 140
Microbial biosensors 140
Optical biosensors 141
Surface plasmon resonance technology 141
Label-free optical biosensor 142
Microsensors using with nano/microelectronic communications technology 142
Electrochemical sensors 142
Enzyme electrodes for biosensing 142
Conductometric sensors 143
Electrochemical genosensors 143
Electrochemical nanobiosensor 143
Bioelectronic sensors 144
Phototransistor biochip biosensor 144
Ribozyme-based sensors 144
RiboReporters 145
Concluding remarks and future prospects of biosensor technology 145
Companies developing biosensors for molecular diagnostics 146
Molecular labels and detection 147
Detection technologies for molecular labels 148
Fluorescence and chemiluminescence 148
Fluorescence technologies for label detection 149
Companies with fluorescence and chemiluminescence products 149
Molecular beacons 150
The Green fluorescent protein 151
Multiophoton detection radioimmunoassay 152
Multi-pixel photon counter 152
Enzyme labels and detection by fluorescence 152
Phase-sensitive flow cytometry 153
Microtransponder-based DNA diagnostics 153
Laboratory Multiple Analyte Profile 154
Multiple labels 154
Protein-DNA chimeras for detection of small numbers of molecules 155
Single molecule detection 155
Atomic force microscopy 156
Capillary electrophoresis 156
Confocal laser scanning 156
nCounter Analysis System 156
Spectrally resolved fluorescence lifetime imaging microscopy 157
Molecular imaging 157
Basic research in molecular imaging 158
Devices for molecular imaging 158
Molecular imaging in clinical practice 158
Challenges and future prospects of molecular imaging 159
Companies involved in molecular imaging 159
Nanobiotechnology for molecular diagnostics 159
Magnetic nanoparticles 160
Gold nanoparticles 161
Quantum dot technology 162
Nanotechnology on a chip 163
Nanogen's NanoChip 163
Fullerene photodetectors for chemiluminescence detection on microfluidic chip 163
Diagnostics based on nanopore technology 164
Nanosensors 164
Detection of cocaine molecules by nanoparticle-labeled aptasensors 164
Nanosensors for glucose monitoring 165
PEBBLE nanosensors 165
Quartz nanobalance biosensor 165
Cantilever arrays 165
Resonance Light Scattering technology 166
DNA nanomachines for molecular diagnostics 167
Nanobarcodes technology for molecular diagnostics 167
Qdot nanobarcode for multiplexed gene expression profiling 167
Role of nanobiotechnology in improving molecular diagnostics 168
Companies involved in nanomolecular diagnostics 168
Concluding remarks about nanodiagnostics 171
Future prospects of nanodiagnostics 171
4. Proteomic Technologies for Molecular Diagnostics 173
Introduction 173
Proteomic technologies 173
Biomarkers of disease 173
Proteomic tools for biomarkers 173
Search for biomarkers in body fluids 174
Captamers with proximity extension assay for proteins 174
Cyclical amplification of proteins 174
Detection of misfolded proteins by ELISA with exponential signal amplification 175
Diagnostics based on designed repeat proteins 175
Differential Peptide Display 175
Light-switching excimer probes 176
MALDI-TOF MS 176
Molecular beacon aptamer 177
Molecular beacon assay 177
Proteomic patterns 178
Real-time PCR for protein quantification 178
Protein biochip technologies 179
ProteinChip 180
LabChip for protein analysis 180
TRINECTIN proteome chip 181
Protein chips for antigen-antibody interactions molecular diagnostics 181
Microfluidic devices for proteomics-based diagnostics 181
Nanotechnology-based protein biochips/microarrays 182
Nanoparticle protein chip 182
Protein nanobiochip 182
Protein biochips based on fluorescence planar wave guide technology 182
New developments in protein chips/microarrays 183
Antibody microarrays 183
Aptamer-based protein biochip 184
Multiplexed Protein Profiling on Microarrays 184
Proteomic pattern analysis 184
Single molecule array 185
Viral protein chip 185
Commercial development of protein chips for molecular diagnostics 185
Proteome Identification Kit 187
Laser capture microdissection (LCM) 187
LCM technology 187
Applications of LCM in molecular diagnostics 188
Proteomic diagnosis of CNS disorders 188
Cerebrospinal fluids tests based on proteomics 188
Urine tests for CNS disorders based on proteins in urine 189
Diagnosis of CNS disorders by examination of proteins in the blood 189
Diagnosis of CNS disorders by examination of proteins in tears 190
Role of proteomics in the diagnosis of Alzheimer's disease 190
Role of proteomics in the diagnosis of Creutzfeldt-Jakob disease 191
Future prospects of use of proteomics for diagnosis of CNS disorders 191
Concluding remarks on the use of proteomics in diagnostics 191
5. Molecular Diagnosis of Genetic Disorders 193
Introduction 193
Cytogenetics 194
FISH with probes to the telomeres 194
Single copy FISH probes 194
Comparative genomic hybridization 195
Use of biochips in genetic disorders 195
Representational oligonucleotide microarray analysis 196
SignatureChip®-based diagnostics for cytogenetic abnormalities 196
Diagnosis of genomic rearrangements by multiplex PCR 196
Quantitative fluorescent PCR 196
Mutation detection technologies 197
PCR-based methods for mutation detection 198
Cleavase Fragment Length Polymorphism 198
Direct dideoxy DNA sequencing 198
Digital Genetic Analysis (DGA) 198
Fluorescence-based directed termination PCR 199
Heteroduplex analysis 199
Restriction fragment length polymorphism 200
Single-stranded conformation polymorphism (SSCP) analysis 200
TaqMan real-time PCR 201
Non-PCR methods for mutation detection 201
Arrayed primer extension 201
BEAMing (beads, emulsion, amplification, and magnetics) 201
ELISA-protein truncation test 202
Enzymatic mutation detection 202
Specific anchor nucleotide incorporation 202
Conversion analysis for mutation detection 203
Biochip technologies for mutation detection 203
Combination of FISH and gene chips 204
Haplotype Specific Extraction 204
Technologies for SNP analysis 204
DNA sequencing 205
Electrochemical DNA probes 206
Use of NanoChip for detection of SNPs 206
Single base extension-tag array 206
Laboratory Multiple Analyte Profile 207
SNP genotyping with gold nanoparticle probes 207
PCR-CTPP (confronting two-pair primers) 207
Peptide nucleic acid probes for SNP detection 208
SNP genotyping on a genome-wide amplified DOP-PCR template 208
Pyrosequencing 208
Reversed enzyme activity DNA interrogation test 209
Smart amplification process version 2 209
Zinc finger proteins 210
UCAN method (Takara Biomedical) 210
Biochip and microarray-based detection of SNPs 210
SNP genotyping by MassARRAY 210
Electronic dot blot assay 211
Biochip combining BeadArray and ZipCode technologies 211
SNP-IT primer-extension technology 211
OmniScan SNP genotyping 212
Affymetrix SNP genotyping array 212
Concluding remarks on SNP genotyping 212
Limitations of SNP in genetic testing 213
Haplotyping versus SNP genotyping 213
Nanofluidics technology for high throughput SNP genotyping 213
Companies involved in developing technologies/products for SNP analysis 214
Role of copy number variations in genetic diagnostic testing 215
CNVs in various diseases 215
CNVs in genetic epilepsy syndromes 215
CNVs associated with schizophrenia 216
Methods for determination of CNVs 216
Digital array for CNV detection 216
Wellcome Trust Case Control Consortium CNV typing array 217
CNVer algorithm for CNV detection 217
Study of rare variants in pinpointing disease-causing genes 217
Prenatal DNA diagnosis 218
Amniocentesis 218
Chorionic villus sampling 218
Separating fetal cells in maternal blood for genetic diagnosis 218
Antenatal screening for Down's syndrome 219
Fetal DNA in maternal blood 219