Molecular Diagnostics

Part I: Technologies & Applications

by
Prof. K. K. Jain
MD, FRACS, FFPM

Jain PharmaBiotech
Basel, Switzerland

July 2012

A Jain PharmaBiotech Report

AUTHOR'S

BIOGRAPHY

Professor K. K. Jain is a neurologist/neurosurgeon by training and has been working in

the biotechnology/biopharmaceuticals industry for several years. He received graduate
training in both Europe and USA, has held academic positions in several countries, and is
a Fellow of the Faculty of Pharmaceutical Medicine of the Royal College of Physicians of
UK.
Prof. Jain is the author of 440 publications including 21 books (4 as editor) and 50 special
reports, which have covered important areas in biotechnology, gene therapy and
biopharmaceuticals. His books include "Role of Nanobiotechnology in Molecular
Diagnostics" (2006), "Handbook of Nanomedicine" (Humana/Springer 2008; Chinese
edition, Peking University Press 2011, 2nd ed Springer 2012), Textbook of Personalized
Medicine (Springer 2009), "Handbook of Biomarkers" (Springer 2010), Applications of
Biotechnology in Cardiovascular Therapeutics (Springer 2011), and Applications of
Biotechnology in Neurology (Springer 2012).

ABOUT

THIS

REPORT

Prof. Jain wrote the first commercial report on DNA Diagnostics in 1995, which was
published by PJB Publication, London. This was updated in 1997 as Molecular
Diagnostics I and the next edition, Molecular Diagnostics II, was published in 1999 both
by Decision Resources Inc, USA. All the three versions of the reports were well accepted
and sold widely. The current version was originally published by Jain PharmaBiotech in
2001 and is constantly updated and revised since then. Not only was this the first such
report on molecular diagnostics, it is the longest continuously published report on this
topic (17 years). It is also the most comprehensive and detailed report on this topic
containing profiles of the largest number of companies involved in molecular diagnostics.

July 2012 (continuously published since 1995)

Copyright 2012 by
Jain PharmaBiotech
Blsiring 7
CH-4057 Basel
Switzerland
Tel & Fax:
Email:
Web site:

+4161-6924461
info@pharmabiotech.ch
http://pharmabiotech.ch/

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, or otherwise without the prior written
permission of the Publisher. This report may not be lent, resold or otherwise traded in any manner without the
consent of the Publisher. While all reasonable steps have been taken to ensure the accuracy of the information
presented, the Publisher cannot accept responsibility for inadvertent errors or omissions.

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CONTENTS
0. Executive Summary .............................................................................. 24
1. Introduction ......................................................................................... 26
2. Molecular Diagnostic Technologies ....................................................... 50
3. Biochips, Biosensors, and Molecular Labels ........................................ 128
4. Proteomic Technologies for Molecular Diagnostics ............................. 180
5. Molecular Diagnosis of Genetic Disorders ........................................... 200
6. Molecular diagnosis of cardiovascular disorders ................................. 262
7. Molecular Diagnosis of Infections ....................................................... 270
8. Molecular Diagnosis of Cancer ............................................................ 350
9. Molecular Diagnostics in Biopharmaceutical Industry & Healthcare ... 438
10. Molecular Diagnostics in Forensic Medicine and Biological Warfare .. 494
11. References ........................................................................................ 518

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TABLE

OF

CONTENTS

0. Executive Summary .............................................................................. 24

1. Introduction ......................................................................................... 26
Definitions and scope of the subject .................................................................................. 26
Historical evolution of molecular diagnostics ..................................................................... 26
Molecular biology relevant to molecular diagnostics .......................................................... 27
DNA ............................................................................................................................... 27
DNA polymerases ........................................................................................................ 27
Restriction endonucleases ............................................................................................ 28
DNA methylation ......................................................................................................... 28
RNA ............................................................................................................................... 29
RNA polymerases ........................................................................................................ 29
Non-coding RNAs ........................................................................................................ 29
DNA transcription ............................................................................................................. 30
Chromosomes ................................................................................................................. 30
Telomeres ....................................................................................................................... 31
Mitochondrial DNA............................................................................................................ 31
Genes............................................................................................................................. 32
The genetic code ......................................................................................................... 32
Gene expression ......................................................................................................... 32
The human genome ......................................................................................................... 33
Variations in the human genome ........................................................................................ 33
Variations in DNA sequences ........................................................................................ 33
Single nucleotide polymorphisms .................................................................................. 34
Haplotyping ..................................................................................................................... 34
Copy number variations in the human genome ............................................................... 35
Genotype and haplotypes ............................................................................................. 36
Insertions and deletions in the human genome ............................................................... 36
Complex chromosomal rearrangements ......................................................................... 37
Large scale variation in human genome ......................................................................... 37
Structural variations in the human genome .................................................................... 38
Replication of the DNA helix ......................................................................................... 38
Transposons ............................................................................................................... 39
Proteins .......................................................................................................................... 39
Proteomics ...................................................................................................................... 39
Monoclonal antibodies ...................................................................................................... 40
Aptamers ........................................................................................................................ 40
Basics of molecular diagnostics ......................................................................................... 40
Tracking DNA: the Southern blot........................................................................................ 40
Pulsed-field gel electrophoresis .......................................................................................... 41
DNA Probes ..................................................................................................................... 41
The polymerase chain reaction .......................................................................................... 42
Basic Principles of PCR ................................................................................................. 42
Target selection .......................................................................................................... 42
Detection of amplified DNA ........................................................................................... 42
Impact of human genome project on molecular diagnostics .............................................. 43
Mapping and sequencing of structural variation from human genomes ............................... 44
1000 Genomes Project...................................................................................................... 44
Human Variome Project ............................................................................................... 45
Role of bioinformatics in molecular diagnostics ................................................................. 46
Systems biology approach to molecular diagnostics .......................................................... 46
Biomarkers ........................................................................................................................ 46
Applications of molecular diagnostics ................................................................................ 47

2. Molecular Diagnostic Technologies ....................................................... 50

Introduction....................................................................................................................... 50
DNA sample collection and extraction ................................................................................ 50
Blood samples ................................................................................................................. 50
Buccal swabs and saliva .................................................................................................... 50
Dried blood spots ............................................................................................................. 51
Formalin-fixed, paraffin-embedded tissues .......................................................................... 51
Manual vs automated DNA extraction ................................................................................. 52
Urine samples for transrenal DNA ...................................................................................... 52
Sample preparation ........................................................................................................... 53
Pressure Cycling Technology ............................................................................................. 53
Membrane immobilization of nucleic acids ......................................................................... 53
Automation of sample preparation in molecular diagnostics .............................................. 54

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ABI PRISM 6700 Automated Nucleic Acid Workstation ........................................................... 54

BioRobot technology ......................................................................................................... 54
COBAS AmpliPrep System ................................................................................................. 54
GENESIS FE500 Workcell .................................................................................................. 54
GeneMole ........................................................................................................................ 55
PCR BioCube ................................................................................................................... 55
QIAsymphony .................................................................................................................. 55
Tigris instrument system................................................................................................... 55
Techniques for sample preparation that are suitable for automation ....................................... 56
Xtra Amp Genomic DNA Extraction ................................................................................ 56
Extraction of DNA from paraffin sections ........................................................................ 56
Dynabead technology .................................................................................................. 56
SamPrep .................................................................................................................... 56
Use of magnetic particles for automation in genome analysis................................................. 57
Companies involved in nucleic acid isolation ........................................................................ 57
Labeling and detection of nucleic acids .............................................................................. 58
Novel PCR methods ............................................................................................................ 59
Addressing limitations of PCR ............................................................................................ 59
CAST-PCR ....................................................................................................................... 59
Combined PCR-ELISA ....................................................................................................... 59
Convection PCR ............................................................................................................... 60
Digital PCR ...................................................................................................................... 60
Emulsion PCR .................................................................................................................. 61
ExCyto PCR ..................................................................................................................... 62
Long and accurate PCR ..................................................................................................... 62
Multiplex PCR .................................................................................................................. 62
Overlap extension PCR...................................................................................................... 62
Real-time PCR systems ..................................................................................................... 63
Dyes used in real-time PCR .......................................................................................... 63
Commercially available real-time PCR systems................................................................ 64
LightCycler PCR system ............................................................................................... 65
LightUp probes based on real-time PCR ......................................................................... 65
READ real-time PCR method ...................................................................................... 65
StellARray technology ............................................................................................... 66
7500 Fast Dx Real-Time PCR Instrument........................................................................ 66
Applications of real-time PCR ........................................................................................ 66
Limitations of real-time PCR ......................................................................................... 67
Quantitative PCR for accurate low level DNA analysis ....................................................... 67
qPCR for quantification of circulating mtDNA................................................................... 67
Guidelines for real-time qPCR ....................................................................................... 68
Future applications of real-time qPCR ............................................................................ 68
Reverse transcriptase (RT)-PCR ......................................................................................... 68
Standardized reverse transcriptase PCR ......................................................................... 69
Single cell PCR ................................................................................................................. 70
LATE-PCR ................................................................................................................... 70
COLD-PCR .................................................................................................................. 70
AmpliGrid-System ....................................................................................................... 71
DNA melt analysis .............................................................................................................. 71
High-resolution DNA melt analysis for genotyping ................................................................ 71
PCR device for DNA melt analysis in space .......................................................................... 72
Monitoring of gene amplification in molecular diagnostics................................................. 72
Non-PCR nucleic acid amplification methods ..................................................................... 72
DNA probes with conjugated minor groove binder ................................................................ 73
Dynamic Flux Amplification ............................................................................................... 73
Isothermal nucleic acid amplifications ................................................................................. 73
Isothermal reaction for amplification of oligonucleotides .................................................. 73
Isothermal and chimeric primer-initiated amplification of nucleic acids ............................... 74
Loop-mediated amplification ......................................................................................... 74
Linked Linear Amplification ................................................................................................ 74
Multiplex Ligation-Dependent Probe Amplification ................................................................ 75
Rapid analysis of gene expression ...................................................................................... 75
Rolling circle amplification technology ................................................................................. 75
Gene-based diagnostics through RCAT ........................................................................... 76
RCAT-immunodiagnostics ............................................................................................. 77
RCAT-pharmacogenomics ............................................................................................. 77
Circle-to-circle amplification ......................................................................................... 77
Ramification amplification method ...................................................................................... 77
Single Primer Isothermal Amplification................................................................................ 78
Transcription mediated amplification .................................................................................. 78
WAVE nucleic acid fragment analysis system ....................................................................... 78
Technologies for signal amplification ................................................................................. 79

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3 DNA dendrimer signal amplification ................................................................................. 79

Hybridization signal amplification method............................................................................ 80
Signal mediated amplification of RNA technology ................................................................. 81
Invader assays ................................................................................................................ 82
Hybrid Capture technology ................................................................................................ 83
Branched DNA test ........................................................................................................... 84
Tyramide signal amplification ............................................................................................ 84
Non-enzymatic signal amplification technologies .................................................................. 84
Direct molecular analysis without amplification ................................................................ 85
Trilogy Platform ............................................................................................................ 85
Direct detection of dsDNA ................................................................................................. 86
Multiplex assays ................................................................................................................ 87
Fluorescent in situ hybridization ........................................................................................ 87
FISH technique ................................................................................................................ 88
Applications of FISH ......................................................................................................... 88
Modifications of FISH ........................................................................................................ 89
Direct visual in situ hybridization ....................................................................................... 89
Direct labeled Satellite FISH probes .................................................................................... 89
Comparative genomic hybridization .................................................................................... 90
Primed in situ labeling ...................................................................................................... 90
Interphase FISH .............................................................................................................. 90
FISH with telomere-specific probes .................................................................................... 91
Multicolor FISH ................................................................................................................ 91
Simultaneous Ultrasensitive Subpopulation staining/Hybridization In situ ........................... 91
Automation of FISH .......................................................................................................... 92
Companies involved in FISH diagnostics .............................................................................. 92
RNA diagnostics ................................................................................................................. 93
RNA isolation from tissue samples ...................................................................................... 93
Commercially available tests for mRNA detection and quantitation ......................................... 93
Branched-chain DNA assay for measurement of RNA ............................................................ 94
Cycling probe technology .................................................................................................. 94
Invader RNA assays ......................................................................................................... 95
Linear RNA amplification ................................................................................................... 95
Non-isotopic RNase cleavage assay .................................................................................... 95
Nucleic acid sequence-based amplification .......................................................................... 96
Q Beta replicase system.................................................................................................... 98
RNAScope ....................................................................................................................... 98
RNA expression profiling ................................................................................................... 98
Visualization of mRNA expression in vivo........................................................................ 98
Solid Phase Transcription Chain Reaction ............................................................................ 99
Transcriptome analysis ..................................................................................................... 99
MicroRNA diagnostics ....................................................................................................... 99
Microarray vs quantitative PCR for measuring miRNAs ................................................... 100
Microarrays for analysis of miRNA gene expression........................................................ 100
miR-TRAP to identify miRNA targets in vivo .................................................................. 101
Modification of in situ hybridization for detection of miRNAs ........................................... 101
Nuclease Protection Assay to measure miRNA expression ............................................... 102
Real-time PCR for expression profiling of miRNAs .......................................................... 102
Use of LNA to explore miRNA ...................................................................................... 102
Whole genome amplification ............................................................................................ 103
Companies that provide technologies for whole genome amplification ................................... 103
QIAGENs Repli-G system ........................................................................................... 103
GenomePlex Whole Genome Amplification ................................................................. 104
DNA sequencing ............................................................................................................... 104
Companies involved in sequencing ................................................................................... 106
Applications of next generation sequencing in molecular diagnostics..................................... 108
Companies developing sequencing for molecular diagnostics ............................................... 109
Genome-wide approach for chromatin mapping .............................................................. 110
Mitochondrial sequencing ................................................................................................ 110
Identification of unknown DNA sequences ......................................................................... 110
Optical mapping ............................................................................................................... 111
Gene expression analysis ................................................................................................. 111
Gene expression profiling on whole blood samples ............................................................. 112
Gene expression patterns of white blood cells .................................................................... 112
Gene expression profiling based on alternative RNA splicing ................................................ 113
MAUI (MicroArray User Interface) hybridization ................................................................. 113
Monitoring in vivo gene expression by molecular imaging.................................................... 114
Serial analysis of gene expression (SAGE) ......................................................................... 114
Single-cell gene expression analysis ................................................................................. 114
T cell receptor expression analysis ................................................................................... 115
Tangerine expression profiling ...................................................................................... 115

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Whole genome expression array ...................................................................................... 116

Ziplex system ............................................................................................................. 116
Companies involved in gene expression analysis ................................................................ 117
Peptide nucleic acid technology ....................................................................................... 118
Use of PNA with fluorescence in situ hybridization .............................................................. 118
PNA and PCR ................................................................................................................. 119
Use of PNA with biosensors ............................................................................................. 119
PNA-based PD-loop technology ........................................................................................ 120
PNA-DNA hybrid quadruplexes ......................................................................................... 120
Companies involved in PNA diagnostics ............................................................................. 120
Locked nucleic acids ........................................................................................................ 121
Zip Nucleic Acids .............................................................................................................. 121
Electrochemical detection of DNA .................................................................................... 122
Mediated nucleic acid oxidation ........................................................................................ 122
Detection of hybridized nucleic acid with cyclic voltametry .................................................. 123
Electrochemical detection based on Toshibas CMOS technology........................................... 123
Concluding remarks on electrochemical DNA detection ....................................................... 123
Bead-based assay platforms ............................................................................................ 124
Scorpions technology .................................................................................................... 125
The Scorpions reaction ................................................................................................... 125
Applications of Scorpions ................................................................................................ 125
Nucleic acid lateral flow molecular diagnostics ................................................................ 126
Omics-based tests ........................................................................................................... 127

3. Biochips, Biosensors, and Nanobiotechnology .................................... 128

Introduction to biochip technology .................................................................................. 128
Applications of biochips in diagnostics............................................................................. 128
GeneChip ...................................................................................................................... 129
GeneChip Human Genome Arrays ............................................................................... 130
AmpliChip CYP450 ..................................................................................................... 130
Electronic detection of nucleic acids on microarrays ............................................................ 130
Microchip capillary electrophoresis ................................................................................... 131
Strand displacement amplification on a biochip .................................................................. 131
Rolling circle amplification on microarrays ......................................................................... 131
LiquiChip-RCAT ......................................................................................................... 131
Fast PCR biochip ............................................................................................................ 132
Multiplex microarray-enhanced PCR for DNA analysis ......................................................... 132
Multiplexed Molecular Profiling ......................................................................................... 132
Universal DNA microarray combining PCR and ligase detection reaction ................................ 133
Genomewide association scans ........................................................................................ 133
Whole genome chips/microarrays..................................................................................... 134
Transposon insertion site profiling chip ............................................................................. 134
Standardizing the microarrays ......................................................................................... 135
Companies involved in developing biochip technology for diagnostics ................................... 135
Future of biochip technology for molecular diagnostics ........................................................ 136
Microfluidic chips ............................................................................................................. 137
Fish-on-chip .................................................................................................................. 137
Lab-on-a-chip ................................................................................................................ 137
LabCD .......................................................................................................................... 138
Micronics' microfluidic technology..................................................................................... 138
Microfluidic chips/arrays using PCR................................................................................... 138
Microfluidic automated DNA analysis using PCR............................................................. 138
Digital PCR Array ...................................................................................................... 138
Digital PCR on a SlipChip............................................................................................ 139
Microfluidic chips integrated with RCAT ............................................................................. 139
Microfluidic chips integrated with PET ............................................................................... 139
Companies developing microfluidic technologies ................................................................ 140
Biosensor technologies .................................................................................................... 141
Classification of biosensor technologies ............................................................................. 141
DNA-based biosensors .................................................................................................... 142
DNA hybridization biosensor chips ............................................................................... 142
PCR-free DNA biosensor ............................................................................................. 142
DNA based biosensor to detects metallic ions ............................................................... 143
Genetically engineered B lymphocytes .............................................................................. 143
Biosensors immunoassays ............................................................................................... 143
PNA (peptide nucleic acid)-based biosensors ..................................................................... 144
Protein-based biosensors ................................................................................................ 144
Antibody biosensors ....................................................................................................... 144
Cell-based biosensors (cytosensors) ................................................................................. 144
Multicell biosensors ................................................................................................... 145
Microbial biosensors .................................................................................................. 145

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Optical biosensors .......................................................................................................... 146

Surface plasmon resonance technology ........................................................................ 146
Label-free optical biosensor ........................................................................................ 147
Microsensors using with nano/microelectronic communications technology ............................ 147
Electrochemical sensors .................................................................................................. 147
Enzyme electrodes for biosensing................................................................................ 148
Conductometric sensors ............................................................................................. 148
Electrochemical genosensors ...................................................................................... 148
Electrochemical nanobiosensor ................................................................................... 148
Bioelectronic sensors ...................................................................................................... 149
Phototransistor biochip biosensor ..................................................................................... 149
Ribozyme-based sensors ................................................................................................. 150
RiboReporters ........................................................................................................... 150
Concluding remarks and future prospects of biosensor technology ....................................... 151
Companies developing biosensors for molecular diagnostics ................................................ 151
Molecular labels and detection ......................................................................................... 152
Detection technologies for molecular labels ....................................................................... 153
Fluorescence and chemiluminescence ............................................................................... 153
Fluorescence technologies for label detection ................................................................ 154
Companies with fluorescence and chemiluminescence products ...................................... 154
Molecular beacons .......................................................................................................... 155
The Green fluorescent protein.......................................................................................... 156
Multiophoton detection radioimmunoassay ........................................................................ 157
Multi-pixel photon counter............................................................................................... 157
Enzyme labels and detection by fluorescence..................................................................... 157
Phase-sensitive flow cytometry ........................................................................................ 158
Microtransponder-based DNA diagnostics .......................................................................... 158
Laboratory Multiple Analyte Profile ................................................................................... 159
Multiple labels ............................................................................................................... 159
Protein-DNA chimeras for detection of small numbers of molecules ................................ 160
Single molecule detection ................................................................................................ 160
Atomic force microscopy ................................................................................................. 161
Capillary electrophoresis ................................................................................................. 161
Confocal laser scanning .................................................................................................. 161
Time domain optical imaging technology ........................................................................... 161
nCounter Analysis System ............................................................................................... 162
Spectrally resolved fluorescence lifetime imaging microscopy .............................................. 162
Molecular imaging............................................................................................................ 162
Technologies for molecular imaging .................................................................................. 162
Basic research in molecular imaging ................................................................................. 164
Devices for molecular imaging ......................................................................................... 164
Molecular imaging in clinical practice ................................................................................ 164
Challenges and future prospects of molecular imaging ........................................................ 165
Companies involved in molecular imaging ......................................................................... 165
Nanobiotechnology for molecular diagnostics .................................................................. 165
Magnetic nanoparticles ................................................................................................... 166
Gold nanoparticles ......................................................................................................... 167
Immunoliposome-PCR .................................................................................................... 168
Quantum dot technology ................................................................................................. 168
Nanotechnology on a chip ............................................................................................... 169
Nanogen's NanoChip ...................................................................................................... 170
Fullerene photodetectors for chemiluminescence detection on microfluidic chip ...................... 170
Diagnostics based on nanopore technology ....................................................................... 170
Nanosensors ................................................................................................................. 170
Detection of cocaine molecules by nanoparticle-labeled aptasensors ................................ 171
Nanosensors for glucose monitoring ............................................................................ 171
PEBBLE nanosensors ................................................................................................. 171
Quartz nanobalance biosensor .................................................................................... 172
Cantilever arrays ........................................................................................................... 172
Resonance Light Scattering technology ............................................................................. 172
DNA nanomachines for molecular diagnostics .................................................................... 173
Nanobarcodes technology for molecular diagnostics ........................................................... 174
Qdot nanobarcode for multiplexed gene expression profiling................................................ 174
Role of nanobiotechnology in improving molecular diagnostics ............................................. 174
Companies involved in nanomolecular diagnostics .............................................................. 175
Concluding remarks about nanodiagnostics ....................................................................... 177
Future prospects of nanodiagnostics ................................................................................. 178

4. Proteomic Technologies for Molecular Diagnostics ............................. 180

Introduction..................................................................................................................... 180
Proteomic technologies.................................................................................................... 180

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Biomarkers of disease .................................................................................................... 180

Proteomic tools for biomarkers ................................................................................... 180
Search for biomarkers in body fluids ............................................................................ 181
Captamers with proximity extension assay for proteins ....................................................... 181
Cyclical amplification of proteins ...................................................................................... 181
Detection of misfolded proteins by ELISA with exponential signal amplification ...................... 182
Detection of proteins by Western blot ............................................................................... 182
Diagnostics based on designed repeat proteins .................................................................. 182
Differential Peptide Display ............................................................................................. 183
Light-switching excimer probes ........................................................................................ 183
MALDI-TOF MS .............................................................................................................. 184
Molecular beacon aptamer .............................................................................................. 184
Molecular beacon assay .................................................................................................. 185
Proteomic patterns ......................................................................................................... 185
Real-time PCR for protein quantification ............................................................................ 186
Protein biochip technologies ............................................................................................ 186
ProteinChip ................................................................................................................... 187
LabChip for protein analysis ............................................................................................ 188
TRINECTIN proteome chip ............................................................................................... 188
Protein chips for antigen-antibody interactions molecular diagnostics ................................... 188
Microfluidic devices for proteomics-based diagnostics ......................................................... 189
Nanotechnology-based protein biochips/microarrays .......................................................... 189
Nanoparticle protein chip ........................................................................................... 189
Protein nanobiochip ................................................................................................... 189
Protein biochips based on fluorescence planar wave guide technology ............................. 190
New developments in protein chips/microarrays ................................................................ 190
Antibody microarrays................................................................................................. 191
Aptamer-based protein biochip ................................................................................... 191
Multiplexed Protein Profiling on Microarrays .................................................................. 191
Proteomic pattern analysis ......................................................................................... 192
Single molecule array ................................................................................................ 192
Viral protein chip....................................................................................................... 192
Commercial development of protein chips for molecular diagnostics ..................................... 193
Proteome Identification Kit .............................................................................................. 194
Laser capture microdissection (LCM) ............................................................................... 194
LCM technology ............................................................................................................. 194
Applications of LCM in molecular diagnostics...................................................................... 195
Proteomic diagnosis of CNS disorders .............................................................................. 195
Cerebrospinal fluids tests based on proteomics .................................................................. 195
Urine tests for CNS disorders based on proteins in urine ..................................................... 196
Diagnosis of CNS disorders by examination of proteins in the blood...................................... 197
Diagnosis of CNS disorders by examination of proteins in tears ............................................ 197
Role of proteomics in the diagnosis of Alzheimer's disease .................................................. 198
Role of proteomics in the diagnosis of Creutzfeldt-Jakob disease .......................................... 198
Future prospects of use of proteomics for diagnosis of CNS disorders ................................... 198
Concluding remarks on the use of proteomics in diagnostics ........................................... 199

5. Molecular Diagnosis of Genetic Disorders ........................................... 200

Introduction..................................................................................................................... 200
Cytogenetics .................................................................................................................... 201
FISH with probes to the telomeres ................................................................................... 201
Single copy FISH probes ................................................................................................. 201
Comparative genomic hybridization .................................................................................. 202
Use of biochips in genetic disorders ................................................................................. 202
Representational oligonucleotide microarray analysis .......................................................... 203
SignatureChip-based diagnostics for cytogenetic abnormalities .......................................... 203
Diagnosis of genomic rearrangements by multiplex PCR ................................................. 203
Quantitative fluorescent PCR ........................................................................................... 203
Mutation detection technologies ...................................................................................... 204
PCR-based methods for mutation detection ....................................................................... 205
Cleavase Fragment Length Polymorphism .................................................................... 205
Direct dideoxy DNA sequencing................................................................................... 205
Digital Genetic Analysis (DGA) .................................................................................... 205
Fluorescence-based directed termination PCR ............................................................... 206
Fluorescence melting curve analysis for multiplex mutation detection .............................. 206
Heteroduplex analysis ................................................................................................ 207
Restriction fragment length polymorphism ................................................................... 207
Single-stranded conformation polymorphism (SSCP) analysis ......................................... 207
TaqMan real-time PCR ............................................................................................... 208
Non-PCR methods for mutation detection .......................................................................... 208
Arrayed primer extension ........................................................................................... 208

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BEAMing (beads, emulsion, amplification, and magnetics) .............................................. 209

ELISA-protein truncation test...................................................................................... 209
Enzymatic mutation detection ..................................................................................... 209
Specific anchor nucleotide incorporation....................................................................... 210
Conversion analysis for mutation detection................................................................... 210
Biochip technologies for mutation detection ....................................................................... 210
Combination of FISH and gene chips ........................................................................... 211
Haplotype Specific Extraction ...................................................................................... 211
Use of biosensors for detection of mutations ..................................................................... 211
Technologies for SNP analysis ......................................................................................... 212
DNA sequencing............................................................................................................. 213
Electrochemical DNA probes ............................................................................................ 213
Use of NanoChip for detection of SNPs ......................................................................... 214
Single base extension-tag array ....................................................................................... 214
Laboratory Multiple Analyte Profile ................................................................................... 214
SNP genotyping with gold nanoparticle probes ................................................................... 215
PCR-CTPP (confronting two-pair primers) .......................................................................... 215
Peptide nucleic acid probes for SNP detection .................................................................... 215
SNP genotyping on a genome-wide amplified DOP-PCR template ......................................... 215
Pyrosequencing ............................................................................................................. 216
Reversed enzyme activity DNA interrogation test ............................................................... 216
Smart amplification process version 2............................................................................... 217
Zinc finger proteins ........................................................................................................ 217
UCAN method (Takara Biomedical) ................................................................................... 217
Biochip and microarray-based detection of SNPs ............................................................. 218
SNP genotyping by MassARRAY........................................................................................ 218
Electronic dot blot assay ................................................................................................. 218
Biochip combining BeadArray and ZipCode technologies ..................................................... 219
SNP-IT primer-extension technology ................................................................................ 219
OmniScan SNP genotyping .............................................................................................. 219
Affymetrix SNP genotyping array ..................................................................................... 219
Concluding remarks on SNP genotyping ............................................................................ 220
Limitations of SNP in genetic testing ................................................................................. 220
Haplotyping versus SNP genotyping ................................................................................. 221
Nanofluidics technology for high throughput SNP genotyping ............................................... 221
Companies involved in developing technologies/products for SNP analysis ............................ 221
Role of copy number variations in genetic diagnostic testing .......................................... 223
CNVs in various diseases................................................................................................. 223
CNVs in genetic epilepsy syndromes ............................................................................ 223
CNVs associated with schizophrenia............................................................................. 223
Methods for determination of CNVs .................................................................................. 224
Digital array for CNV detection.................................................................................... 224
Wellcome Trust Case Control Consortium CNV typing array ............................................ 224
CNVer algorithm for CNV detection .............................................................................. 224
Study of rare variants in pinpointing disease-causing genes ................................................ 225
Prenatal DNA diagnosis.................................................................................................... 226
Invasive prenatal diagnostic procedures............................................................................ 226
Amniocentesis .......................................................................................................... 226
Chorionic villus sampling ............................................................................................ 226
Molecular methods for prenatal diagnosis .......................................................................... 226
aCGH for prenatal diagnosis ....................................................................................... 226
BAC HD Scan test ..................................................................................................... 227
FISH for prenatal diagnosis ........................................................................................ 227
PCR for prenatal diagnosis.......................................................................................... 227
In vivo gene expression analysis of the living human fetus ............................................. 228
Non-invasive prenatal molecular diagnostic procedures....................................................... 228
Fetal cells separation from maternal blood for genetic diagnosis ..................................... 228
Digital relative mutation dosage in maternal plasma ...................................................... 228
Prenatal testing based on transrenal DNA from urine ..................................................... 229
Tests on fetal DNA in maternal blood ................................................................................ 229
Sequencing-based methods for prenatal diagnosis from maternal DNA ............................ 229
Directed DNA analysis of maternal blood ...................................................................... 230
Applications of prenatal diagnosis .................................................................................... 231
Antenatal screening for Down's syndrome .................................................................... 232
Diagnosis of congenital infections ................................................................................ 232
Diagnosis of eclampsia............................................................................................... 233
Preimplantation genetic diagnosis ................................................................................... 233
Technologies for preimplantation genetic diagnosis (PGD) ................................................... 234
PCR for preimplantation genetic diagnosis .................................................................... 234
FISH for preimplantation genetic diagnosis ................................................................... 234
Microarrays for preimplantation genetic diagnosis ......................................................... 235

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Conditions detected by preimplantation genetic diagnosis ................................................... 235

The future of preimplantation genetic diagnosis ................................................................. 235
Companies involved in prenatal/preimplantation diagnosis ............................................ 236
Cystic fibrosis .................................................................................................................. 237
Detection of CFTR gene mutations ................................................................................... 237
CFTR technologies of various companies ........................................................................... 238
Genzyme's CF gene sequencing .................................................................................. 239
CF Plus Tag-It Cystic Fibrosis Kit .............................................................................. 239
Asuragen's bead array test ......................................................................................... 239
The Ambry CF Test .................................................................................................... 240
Biochip for CF diagnosis ............................................................................................. 240
Identification of CF variants by PCR/Oligonucleotide Ligation Assay ................................. 241
SensiGene (SEQUENOM) CF carrier screening test ......................................................... 241
Serum proteomic signature for CF using antibody microarrays ........................................ 241
Guidelines for genetic screening for CF ............................................................................. 241
Congenital adrenal hyperplasia ........................................................................................ 242
Primary immunodeficiencies ............................................................................................ 242
Hematological disorders .................................................................................................. 243
Hemoglobinopathies ....................................................................................................... 243
Sickle cell anemia ..................................................................................................... 243
Thalassemia ............................................................................................................. 244
Paroxysmal nocturnal hemoglobinuria .......................................................................... 244
Hemophilia .................................................................................................................... 244
Hereditary hemochromatosis ........................................................................................... 245
Polycystic kidney disease................................................................................................. 245
Hereditary metabolic disorders ........................................................................................ 246
Lesch-Nyhan Syndrome .................................................................................................. 246
Gauchers Disease .......................................................................................................... 246
Acute Intermittent Porphyria ........................................................................................... 246
Phenylketonuria ............................................................................................................. 247
Hereditary periodic fever................................................................................................. 247
Achondroplasia ................................................................................................................ 247
Molecular diagnosis of eye diseases................................................................................. 248
Molecular diagnosis of retinitis pigmentosa ........................................................................ 248
Genetic screening for glaucoma ....................................................................................... 248
Role of molecular diagnostics in rheumatoid arthritis ...................................................... 249
Molecular diagnosis of neurogenetic disorders ................................................................ 249
Alzheimer's disease ........................................................................................................ 251
Autism spectrum disorders .............................................................................................. 251
CNVs associated with autism ...................................................................................... 251
Charcot-Marie Tooth disease ........................................................................................... 252
Down syndrome ............................................................................................................. 253
Duchenne and Becker muscular dystrophy ........................................................................ 253
eNOS gene polymorphisms as predictor of cerebral aneurysm rupture .................................. 254
Fragile X syndrome ........................................................................................................ 254
Huntington disease ........................................................................................................ 255
Hereditary neuropathy with liability to pressure palsies ....................................................... 255
Mitochondrial disorders affecting the nervous system ......................................................... 256
Parkinsons disease ........................................................................................................ 256
Pompes disease ............................................................................................................ 257
Spinal muscular atrophy ................................................................................................. 257
Triple repeat disorders .................................................................................................... 257
Genetic testing for disease predisposition ....................................................................... 258
Direct-to-consumer genetic tests ..................................................................................... 258

6. Molecular diagnosis of cardiovascular disorders ................................. 262

Introduction..................................................................................................................... 262
Coronary heart disease .................................................................................................... 263
Genomics of coronary heart disease ................................................................................. 263
Cardiomyopathy ............................................................................................................... 264
Familial Hypertrophic Cardiomyopathy .............................................................................. 264
Idiopathic dilated cardiomyopathy .................................................................................... 264
Cardiac Arrhythmias ........................................................................................................ 264
Long Q-T Syndrome ....................................................................................................... 264
Familial atrial fibrillation .................................................................................................. 265
Idiopathic ventricular fibrillation ....................................................................................... 265
Congestive heart failure ................................................................................................... 265
Hypertension ................................................................................................................... 266
Disturbances of blood lipids ............................................................................................. 266
Familial dyslipoproteinemias ............................................................................................ 266
Hypercholesterolemia ..................................................................................................... 266

- 11 -

Thrombotic disorders ....................................................................................................... 267

Factor V Leiden mutation ................................................................................................ 267
Pulmonary embolism ...................................................................................................... 268
Hereditary thrombophilia ................................................................................................ 268
Molecular diagnostics for monitoring heart transplant rejection ...................................... 268
AlloMap molecular expression testing ............................................................................. 268
Commercial molecular diagnostics for cardiovascular disorders ...................................... 269

7. Molecular Diagnosis of Infections ....................................................... 270

Introduction..................................................................................................................... 270
Molecular techniques for the diagnosis of infections........................................................ 270
Antibody-enhanced microplate hybridization assays ........................................................... 271
Bacteriophage-based methods for detection of bacteria ...................................................... 271
Biochips/microarrays for detection of microorganisms ........................................................ 272
Lawrence Livermore Microbial Detection Array .............................................................. 272
Biosensors for detection of microorganisms ....................................................................... 272
Ibis T5000 Biosensor System ................................................................................... 272
DNA enzyme immunoassay ............................................................................................. 272
DNA biochip/microarray in diagnosis of infections............................................................... 273
DNA-based typing methods ............................................................................................. 274
Restriction fragment length polymorphism analysis ....................................................... 274
Ribotyping................................................................................................................ 275
Random amplified polymorphic DNA ............................................................................ 275
Combinatorial DNA melting assay ................................................................................ 275
Electrochemical detection of pathogens............................................................................. 275
FISH for detection of infections ........................................................................................ 276
Helicase-dependent isothermal amplification for rapid detection of pathogens ....................... 276
High resolution melt analysis for diagnosis of infections ...................................................... 276
Ligase chain reaction ...................................................................................................... 276
Multiplex PCR for detection of infections ............................................................................ 277
Metagenomic pyrosequencing .......................................................................................... 277
Dual priming oligonucleotide for multiplex PCR ............................................................. 278
Multiplex amplified nominal tandem repeat analysis ...................................................... 279
Tests for sepsis .............................................................................................................. 279
LightCycler SeptiFast Test ....................................................................................... 279
NanoDx ................................................................................................................. 280
SeptiCyte Triage assay .............................................................................................. 280
SepsiTest .............................................................................................................. 280
VYOO Sepsis Test ................................................................................................... 280
NASBA for detection of microorganisms ............................................................................ 281
Nucleic acid probes ........................................................................................................ 281
Neutrophil CD11b expression as a diagnostic marker .......................................................... 281
Optical Mapping ............................................................................................................. 282
PNA-FISH for diagnosis of infections ................................................................................. 282
Proteomic technologies for diagnosis of infections .............................................................. 282
Mass spectrometry for microbial identification............................................................... 282
QuantiFERON technology for pre-molecular diagnosis of infections .................................... 283
Quantitative reverse-transcription PCR for bacterial diagnostics ........................................... 284
Rupture event scanning .................................................................................................. 284
Real-time single-molecule imaging of virus particles ........................................................... 284
Single-strand conformational polymorphism ...................................................................... 284
SmartGene platform for identifying pathogens based on genetic sequences........................... 284
Tessera array technology ................................................................................................ 285
Unyvero Solution ........................................................................................................... 285
Applications, advantages and limitations of molecular diagnostics .................................. 285
Molecular diagnostics versus other microbial detection technologies ..................................... 285
Advantages of nucleic acid-based diagnostics in infections .................................................. 286
Drawbacks of nucleic acid-based diagnostics in infections ................................................... 286
Nanotechnology for detection of infectious agents .............................................................. 287
Bacterial infections .......................................................................................................... 287
Mycobacterium tuberculosis ............................................................................................ 288
Conventional diagnosis of tuberculosis ......................................................................... 289
Combined tuberculin testing and ELISpot PLUS assay ........................................................ 289
Microscopic Observation Drug Susceptible Assay for tuberculosis .................................... 289
Molecular diagnostics for tuberculosis .......................................................................... 290
Biomarker-based tests for tuberculosis ........................................................................ 291
Diagnosis of TB in a POC setting ................................................................................. 292
Diagnosis of drug-resistant MTB infection ..................................................................... 292
Xpert MTB/RIF automated molecular test for MTB ......................................................... 293
Diagnosis of other mycobacteria ................................................................................. 293
Chlamydial infections...................................................................................................... 294

- 12 -

Neisseria gonorrhoeae .................................................................................................... 295

Bacteria associated with bacterial vaginosis ....................................................................... 296
Streptococcal infections .................................................................................................. 296
Group B Streptococci ................................................................................................. 296
Streptococcus pyogenes and Streptococcus dysgalactiae ................................................ 296
Pseudomonas aeruginosa ................................................................................................ 297
Helicobacter pylori ......................................................................................................... 297
Lyme disease ................................................................................................................ 297
Mycoplasmas ................................................................................................................. 299
Fungal infections ............................................................................................................. 299
PCR-based tests for fungal infections ................................................................................ 300
DNA barcode marker for fungi ......................................................................................... 300
DNA sequencing for fungal infections ................................................................................ 300
MALDI-TOF MS for diagnosis of fungal infections ................................................................ 301
Aspergillus .................................................................................................................... 301
Candida species ............................................................................................................. 301
Viral infections ................................................................................................................. 302
HIV/AIDS ...................................................................................................................... 303
Diagnosis of HIV ....................................................................................................... 303
Detection of HIV provirus ........................................................................................... 304
Global Surveillance of HIV-1 genetic variations ............................................................. 304
Genotyping for drug-resistance in HIV ......................................................................... 304
Neonatal screening of infants of HIV-positive mothers ................................................... 305
Phenotyping as predictor of drug susceptibility/resistance in HIV .................................... 306
POC testing for HIV ................................................................................................... 307
PCR for resolution of indeterminate Western blot .......................................................... 307
Screening of cadaveric tissue donors ........................................................................... 307
Tests used for quantification of HIV ............................................................................. 307
Conclusions about HIV diagnostics .............................................................................. 308
Hepatitis viruses ............................................................................................................ 309
Hepatitis A virus ....................................................................................................... 309
Hepatitis B virus ....................................................................................................... 309
Hepatitis C virus ....................................................................................................... 310
Detection and quantification of HCV RNA ...................................................................... 310
Quantification of HCV RNA levels as a guide to antiviral therapy ...................................... 311
Electrochemical DNA chip for diagnosis of HCV.............................................................. 311
HCV Genotyping as a guide to therapy ......................................................................... 311
Enteroviruses ................................................................................................................ 312
Adenoviruses ................................................................................................................. 313
Rhinoviruses.................................................................................................................. 313
Herpes viruses ............................................................................................................... 314
Herpes simplex virus ................................................................................................. 314
Genital and neonatal herpes simplex ........................................................................... 314
Human cytomegalovirus infections .............................................................................. 315
Epstein-Barr virus ..................................................................................................... 315
Human papilloma virus ................................................................................................... 315
Molecular diagnostics for HPV ..................................................................................... 316
Detection of encephalitis viruses ...................................................................................... 317
West Nile and St. Louis encephalitis ............................................................................ 317
Venezuelan equine encephalitis virus ........................................................................... 317
Detection of noroviruses ................................................................................................. 317
Detection of dengue virus ............................................................................................... 318
Protozoal infections ......................................................................................................... 318
Amebiasis ..................................................................................................................... 318
Cryptosporidium parvum ................................................................................................. 318
Leishmaniasis ................................................................................................................ 319
Malaria ......................................................................................................................... 319
Neurocysticercosis ......................................................................................................... 319
Pneumocystis carinii ....................................................................................................... 320
Toxoplasmosis ............................................................................................................... 320
Infections of various systems .......................................................................................... 320
CNS infections ............................................................................................................... 320
Molecular diagnosis in bacterial meningitis ................................................................... 321
Molecular diagnosis in herpes simplex encephalitis ........................................................ 321
Diagnosis of transmissible spongiform encephalopathies ................................................ 321
Molecular diagnosis of respiratory viruses ......................................................................... 322
SARS-associated coronavirus ...................................................................................... 323
Influenza viruses....................................................................................................... 324
Avian influenza ......................................................................................................... 326
H1N1 influenza ......................................................................................................... 329
Gastrointestinal infections ............................................................................................... 332

- 13 -

Periodontal infections ..................................................................................................... 333

Diagnosis of urinary infections by a biosensor .................................................................... 334
Role of molecular diagnostics in septicemia ....................................................................... 334
Limitations and needs of diagnostics for infections ......................................................... 335
Differentiation between live and antibiotic-killed bacteria .................................................... 335
Cell-based methods for identifying pathogenic microorganisms ............................................ 336
Cell-based virus assays .............................................................................................. 336
Cell-based detection of host response to infection ......................................................... 336
Role of molecular diagnostics in hospital acquired infections .......................................... 336
Detection of hospital-acquired bacterial infections .............................................................. 337
Detection of methicillin-resistant S. aureus................................................................... 337
Whole-genome sequencing for investigation of MRSA outbreaks...................................... 338
Detection of vancomycin-resistant enterococci .............................................................. 338
Detection of hospital-acquired C. difficile ...................................................................... 339
Integrated device for rapid detection of organisms associated HAI .................................. 340
Bacterial genome sequencing in antimicrobial resistance ................................................ 340
Detection of hospital-acquired viral infections .................................................................... 341
Molecular diagnosis of BK virus ................................................................................... 341
Diagnosis of hospital-acquired rotavirus gastroenteritis .................................................. 341
Molecular diagnostics and the microbiome ...................................................................... 342
Human Microbiome Project .............................................................................................. 342
Application of metagenomics to study of the microbiome .................................................... 342
MicroBiome Analysis Center............................................................................................. 343
Concluding remarks and future prospects of diagnosis of infections ............................... 343
Rapid point-of-care diagnosis of infection .......................................................................... 344
Diagnosis of viruses using protein fingerprinting ................................................................ 346
QIAplex PCR multiplex technology .................................................................................... 346
Companies involved in molecular diagnosis of infectious diseases .................................. 347

8. Molecular Diagnosis of Cancer ............................................................ 350

Introduction..................................................................................................................... 350
Cancer genomics .............................................................................................................. 350
Cancer genes ................................................................................................................ 351
Oncogenes .................................................................................................................... 351
Tumor Suppressor Genes ................................................................................................ 351
p53 ......................................................................................................................... 352
p16 ......................................................................................................................... 353
CNVs in cancer .............................................................................................................. 353
Allele-specific copy number analysis of tumors.............................................................. 354
Viruses and cancer ......................................................................................................... 354
Detecting viral agents in cancer .................................................................................. 355
Conventional cancer diagnosis ......................................................................................... 356
Molecular techniques for cancer diagnosis ....................................................................... 356
Genome analysis at the molecular level ............................................................................ 358
Mutation detection at molecular level ............................................................................... 358
Expression profiling of tumor cells sorted by flow cytometry ................................................ 359
MicroRNA expression profiling for cancer diagnostics .......................................................... 359
Biomarkers in cancer ...................................................................................................... 359
Circulating nucleic acids as potential biomarkers of cancer ............................................. 360
Circulating nucleosomes in serum of cancer patients ..................................................... 360
Detection of DNA methylation ..................................................................................... 360
eTag assay system for cancer biomarkers .................................................................... 362
HAAH as a biomarker for cancer.................................................................................. 363
LigAmp for detection of gene mutations in cancer ......................................................... 363
Mitochondrial DNA as a cancer biomarker ..................................................................... 363
Oncoproteins as biomarkers for cancer ........................................................................ 364
Sequencing-based approaches for detection of cancer biomarkers ................................... 364
Molecular fingerprinting of cancer .................................................................................... 365
Fluorescent in situ hybridization ....................................................................................... 365
Genetic analysis of cancer ............................................................................................... 366
Comparative genomic hybridization in cancer diagnostics ............................................... 366
Loss of heterozygosity ............................................................................................... 366
Digital karyotyping .................................................................................................... 367
Gene expression profiles predict chromosomal instability in tumors ................................. 367
PCR Techniques ............................................................................................................. 367
Realtime quantitative PCR for diagnosis of cancer ......................................................... 368
Cold-PCR ................................................................................................................. 368
Antibody-based diagnosis of cancer .................................................................................. 368
Monoclonal antibodies for diagnosis of cancer ............................................................... 368
Recombinant antibodies as a novel approach to cancer diagnosis .................................... 369
Combined immunological and nucleic acid tests ................................................................. 369

- 14 -

Combination of MAbs and RT-PCR ............................................................................... 369

Immunobead RT-PCR ................................................................................................ 370
Assays for determining susceptibility to cancer .................................................................. 370
Gene expression profiling in cancer .................................................................................. 370
Microarrays for gene expression profiling in cancer ........................................................ 371
Serial analysis of gene expression (SAGE) .................................................................... 371
DNA tags for finding genes expressed in cancer ............................................................ 372
Suppression subtractive hybridization .......................................................................... 372
Cancer tests based on gene expression profiling ........................................................... 372
Measurement of telomerase activity ................................................................................. 373
Detection of circulating tumor cells in blood ...................................................................... 373
Biochips/microfluidics for detection of CTCs .................................................................. 374
CellSearch ................................................................................................................ 374
CellTracks AutoPrep System.................................................................................. 374
CTCscope system for detection of CTCs........................................................................ 375
CTChip .................................................................................................................. 375
Fiber-optic array scanning technology .......................................................................... 375
Lab-on-chip for the isolation and detection of CTCs ....................................................... 376
MagSweeper............................................................................................................. 376
Future prospects of detection of cancer cells in blood..................................................... 376
Epithelial aggregate separation and isolation ..................................................................... 376
Proteomic technologies for the molecular diagnosis of cancer .............................................. 377
Proteomic technologies for tumor biomarkers ............................................................... 377
Affibodies as contrast agents for imaging in cancer ....................................................... 377
Aptamer-based technology for protein signatures of cancer cells ..................................... 378
Aptamer probes for in vivo diagnosis of cancer ............................................................. 378
Aptamers for combined diagnosis and therapeutics of cancer .......................................... 378
Automated image analysis of nuclear protein distribution ............................................... 379
Laser capture microdissection in oncology .................................................................... 379
Layered expression scanning ...................................................................................... 380
Membrane-type serine protease-1 ............................................................................... 380
Survivin and molecular diagnosis of cancer .................................................................. 380
Biochip/microarrays for cancer diagnosis .......................................................................... 381
Role of DNA microarrays in gene expression profiling..................................................... 381
Biochip detection of FHIT gene.................................................................................... 382
Nanobiotechnology for early detection of cancer ................................................................ 382
Detection of nanoparticle self assembly in tumors by MRI .............................................. 382
Differentiation between normal and cancer cells by nanosensors ..................................... 383
Magnetic nanoparticle probes ..................................................................................... 383
Quantum dots for early detection of cancer .................................................................. 383
Molecular imaging of cancer ............................................................................................ 384
In vivo tumor illumination by adenoviral-GFP ................................................................ 384
PET for in vivo molecular diagnosis of cancer ................................................................ 384
Radiolabeled peptide-based targeting probes for cancer imaging ..................................... 385
Xenon-enhanced MRI................................................................................................. 385
Optical systems for in vivo molecular imaging of cancer ................................................. 385
Detection of micrometastases .......................................................................................... 386
Molecular diagnosis of cancers of various organs ............................................................ 386
Brain tumors ................................................................................................................. 386
Molecular diagnostic methods for brain tumors ............................................................. 387
Glioblastoma multiforme ............................................................................................ 387
Circulating microvesicles as biomarkers of glioblastoma ................................................. 388
Combination of neuroimaging and DNA microarray analysis in GBM ................................. 388
Medulloblastoma ....................................................................................................... 388
Multigene predictor of outcome in GBM ........................................................................ 388
Oligodendroglioma .................................................................................................... 389
Advantages and limitations of molecular diagnosis of brain tumors .................................. 389
Breast cancer ................................................................................................................ 389
Breast cancer genes .................................................................................................. 390
Circulating nucleic acid biomarkers of breast cancer ...................................................... 391
Molecular diagnostic tests for breast cancer .................................................................. 391
Mouse ESC-based assays to evaluate mutations in BRCA2 .............................................. 394
Genomic profiles of breast cancer................................................................................ 395
Role of molecular diagnostics in management of breast cancer ....................................... 396
Tests for prognosis of breast cancer ............................................................................ 400
Prediction of recurrence in breast cancer for personalizing therapy .................................. 402
Cervical cancer .............................................................................................................. 403
Colorectal cancer ........................................................................................................... 404
ColoVantage CRC test ................................................................................................ 404
Detection of familial adenomatous polyposis coli ........................................................... 405
Detection of CRC at precancerous state ....................................................................... 405

- 15 -

Detection of circulating tumor cells in colorectal cancer .................................................. 405

Diagnosis of hereditary nonpolyposis colorectal cancer .................................................. 405
Diagnosis of colorectal cancer from DNA in stools .......................................................... 406
Early diagnosis of colorectal cancer from blood samples ................................................. 407
Guanylyl cyclase C tests for colorectal cancer ............................................................... 407
Minimally invasive screening for colorectal cancer ......................................................... 407
Gastric cancer ............................................................................................................... 408
Head and neck cancer .................................................................................................... 408
Nanobiochip sensor technique for analysis of oral cancer biomarkers ............................... 408
ProteinChip for diagnosis of head and neck cancer ........................................................ 409
Hematological malignancies ............................................................................................ 409
Chromosome translocations ....................................................................................... 409
Flow cytometry in diagnosis of leukemia ...................................................................... 409
Gene chip technology ................................................................................................ 409
Hairy-cell leukemia.................................................................................................... 410
Laboratory assessment of leukemia ............................................................................. 410
Molecular probes ....................................................................................................... 411
Minimal residual disease ............................................................................................ 411
Screening of gene mutations in chronic myeloproliferative diseases ................................. 412
Lung cancer .................................................................................................................. 412
Melanoma ..................................................................................................................... 415
Ovarian cancer .............................................................................................................. 415
Mutation of genes ..................................................................................................... 415
Relevance of genetic testing to management of ovarian cancer ....................................... 416
Serum biomarkers for early detection of ovarian cancer ................................................. 416
Biomarkers of ovarian cancer ..................................................................................... 416
Concluding remarks on testing for ovarian cancer ......................................................... 417
Pancreatic cancer ........................................................................................................... 417
Prostate cancer .............................................................................................................. 418
Early detection of prostate cancer recurrence by nanotechnology .................................... 418
Gene expression analysis of prostate cancer ................................................................. 418
Huntingtin Interacting Protein 1 .................................................................................. 419
Integrative genomic and proteomic profiling of prostate cancer....................................... 419
LCM for diagnosis of prostate cancer ........................................................................... 419
PCA3 gene detection in urine ...................................................................................... 420
PCR assay for assessing silencing of protein cadherin 13 gene ........................................ 421
Prolaris test .............................................................................................................. 421
Prostate biopsy for detection of prostatic intraepithelial neoplasia ................................... 421
Prostate Core Mitomic Test ...................................................................................... 421
Screening of multiple SNPs for risk of prostate cancer ................................................... 422
Semen testing for prostate cancer biomarkers .............................................................. 422
Serum-protein fingerprinting in prostate cancer ............................................................ 423
Thyroid cancer ............................................................................................................... 423
Gene expression biomarkers of thyroid cancer .............................................................. 423
Multiple endocrine neoplasia type 2B as risk factor for thyroid cancer .............................. 424
miRNA expression profiling in thyroid cancer ................................................................ 424
Urinary bladder cancer ................................................................................................... 424
Role of molecular diagnostics in the management of cancer ............................................ 425
Risk assessment and prevention of cancer ........................................................................ 425
Role of molecular diagnosis in the design of future cancer therapies ..................................... 426
Molecular classification of cancer ...................................................................................... 426
Determination of cancer prognosis ................................................................................... 426
Prognosis by tumor classification ................................................................................. 426
Prognosis by cancer gene expression ........................................................................... 427
Selection of anticancer drugs based on molecular diagnosis ................................................ 427
Integrated genome-wide analysis of cancer for diagnosis and therapy .................................. 428
Personalized therapy for cancer patients ......................................................................... 428
Pharmacogenetics and cancer therapy .............................................................................. 428
Molecular diagnostics as an aid to selection of cancer therapy ............................................. 429
Drug resistance in cancer ................................................................................................ 429
Role of organizatons in molecular diagnosis of cancer ..................................................... 430
Role of NCI in molecular diagnosis of cancer ...................................................................... 430
Molecular profiling of cancer ....................................................................................... 430
Cancer Genome Atlas ................................................................................................ 430
Cancer Genetic Markers of Susceptibility Project ........................................................... 431
Support for future research in molecular diagnosis of cancer .......................................... 431
Role of the International Cancer Genome Consortium ......................................................... 432
Future prospects of molecular diagnosis of cancer .......................................................... 433
Companies involved in molecular diagnosis of cancer ...................................................... 433

9. Molecular Diagnostics in Biopharmaceutical Industry & Healthcare ... 438

- 16 -

Introduction..................................................................................................................... 438
Molecular diagnostics in biopharmaceutical industry ....................................................... 438
Molecular diagnostic technologies and drug discovery ......................................................... 439
Molecular diagnostics and pharmacogenetics ..................................................................... 439
Molecular toxicology ....................................................................................................... 440
Gene expression studies for toxicology ........................................................................ 441
Toxicogenomics ........................................................................................................ 441
Toxicoproteomics ...................................................................................................... 441
Mitochondrial assays ...................................................................................................... 443
MetaChip/Datachip ......................................................................................................... 443
Molecular diagnostics and pharmacogenomics ................................................................... 444
Molecular diagnostics and therapeutic drug monitoring ....................................................... 445
Applications molecular diagnostics in gene therapy ............................................................ 445
Use of PCR to study biodistribution of gene therapy vectors............................................ 445
PCR for verification of the transcription of DNA ............................................................. 446
In situ PCR for direct quantification of gene transfer into cells ......................................... 446
Detection of retroviruses by reverse transcriptase (RT)-PCR ........................................... 446
Assessment of safety issues of gene transfer ................................................................ 446
Quantitative PCR for monitoring the effectiveness of gene therapy .................................. 446
Use of FISH for analysis of adeno-associated viral vector integration ............................... 447
Monitoring of gene expression by green fluorescent protein ............................................ 447
Quality control of protein therapeutics and vaccines ........................................................... 447
Detection of microbial contamination in biopharmaceutical manufacturing ............................. 448
Role of PCR in detecting contamination ........................................................................ 448
Systems for rapid detection of contaminants ................................................................ 448
Contamination of biopharmaceuticals with prions .......................................................... 449
DNA tagging for control and tracing of drug distribution channels ......................................... 449
Molecular diagnostics for organ transplantation .............................................................. 449
HLA typing .................................................................................................................... 449
Sequencing for HLA typing .............................................................................................. 451
Commercial products for transplant molecular diagnostics ................................................... 451
Post-cardiac transplant patient monitoring for rejection ...................................................... 453
Application of molecular diagnostics in blood transfusion ............................................... 454
Molecular diagnostics for testing transfusion compatibility ................................................... 454
Transmission of infections in blood transfusion .................................................................. 454
Molecular tests for screening of blood supply for viruses ..................................................... 455
Commercial molecular diagnostic technologies for blood screening ....................................... 455
Bridge amplification technology .................................................................................. 456
COBAS AmpliScreen HCV and HIV Assays .................................................................... 456
INACTINE................................................................................................................. 457
NucliSens Extractor system ........................................................................................ 457
Pall's enhanced Bacteria Detection System ................................................................... 457
PCR combined with algorithm method .......................................................................... 458
Prions detection in human blood ................................................................................. 458
PRISM automated system ........................................................................................ 459
Procleix HIV-1/HCV Assay .......................................................................................... 459
West Nile virus detection in human blood ..................................................................... 459
Advantages and limitations of molecular diagnostics for blood screening ............................... 460
Molecular epidemiology ................................................................................................... 460
Molecular epidemiology of genetic diseases ....................................................................... 461
Role of CNVs in study of genetic epidemiology .............................................................. 461
Accumulation of CNVs with aging ................................................................................ 462
Monogenic versus polygenic disorders.......................................................................... 462
Critical issues facing genetic epidemiology ................................................................... 463
Molecular epidemiology of infectious diseases .................................................................... 463
Methods and purposes ............................................................................................... 463
Emerging infections ................................................................................................... 464
Human vs. non-human infections ................................................................................ 464
Genetics and susceptibility to infectious disease ............................................................ 465
Molecular epidemiology of cancer ..................................................................................... 465
Molecular epidemiology of p53 gene mutations ............................................................. 465
Molecular epidemiology of link between virus and cancer ............................................... 466
Molecular epidemiology and cancer prevention ............................................................. 466
SNPs and molecular epidemiology ............................................................................... 466
Molecular diagnostics for identification of food-borne pathogens .................................... 466
Introduction .................................................................................................................. 466
Molecular diagnostic methods used in food-borne infections ................................................ 467
Limitations of use of molecular probes in food analysis ....................................................... 468
Detection of Listeria-contaminated foods .......................................................................... 468
Optical biosensor for detection of Listeria ..................................................................... 468
Real-time PCR for detection of Listeria ......................................................................... 469

- 17 -

Detection of Salmonella .................................................................................................. 469

MicroSEQ Salmonella Detection Kit ........................................................................... 469
E. Coli detection............................................................................................................. 469
MicroSEQ E. Coli Detection Kit ................................................................................. 470
DuPont Bax system ................................................................................................... 470
Detection of rare strains of E. Coli ............................................................................... 470
Companies with technologies for food pathogen detection ................................................... 471
Transmissible spongiform encephalopathies (TSEs) ............................................................ 472
Basis of molecular diagnosis of prion diseases .............................................................. 472
Molecular diagnosis of TSEs ........................................................................................ 473
Companies involved in developing molecular diagnostics for TSEs ................................... 476
Detection of genetically modified food ............................................................................. 476
Molecular diagnostics for detection of doping in sports ................................................... 478
Screening of synthetic glucocorticosteroids in human urine ................................................. 478
Detection of gene doping ................................................................................................ 478
Role of molecular diagnostics in future healthcare .......................................................... 479
Translation of genomic research into genetic testing for healthcare ...................................... 479
Molecular diagnostics and disease management ................................................................. 479
Role of genetic biomarkers in disease management ............................................................ 480
Role of molecular diagnostics in personalized medicine ....................................................... 480
Integrated healthcare ..................................................................................................... 480
Screening................................................................................................................. 481
Early diagnosis ......................................................................................................... 481
Prevention................................................................................................................ 481
Therapy based on molecular diagnosis ......................................................................... 481
Monitoring of therapy ................................................................................................ 482
Advantages and limitations of integrated healthcare ...................................................... 482
Commercially available systems for integrated healthcare .............................................. 483
Combination of diagnostics and therapeutics ..................................................................... 483
Companion diagnostics .............................................................................................. 483
Companies involved in companion diagnostics .............................................................. 483
Point-of-care diagnosis ................................................................................................... 485
Technologies for point-of-care diagnosis ...................................................................... 486
Biochips for POC diagnosis ......................................................................................... 487
Advantages versus disadvantages of POC diagnosis ....................................................... 488
POC Diagnostic Initiative ............................................................................................ 489
Future prospects of POC testing .................................................................................. 490
Companies developing POC diagnosis .......................................................................... 490
The impact of molecular diagnostics on clinical laboratory practice ....................................... 492

10. Molecular Diagnostics in Forensic Medicine and Biological Warfare .. 494

Application of molecular diagnostics in forensic medicine ............................................... 494
Technologies ................................................................................................................. 494
ABO genotyping ........................................................................................................ 494
DNA analysis for identification of ancient or historical specimens ..................................... 495
Extraction of DNA from forensic samples ...................................................................... 495
Fluorescent detection systems .................................................................................... 495
Mitochondrial DNA (mtDNA) analysis ........................................................................... 495
Pressure cycling technology for forensic applications ..................................................... 496
Polymorphic Alu insertions ......................................................................................... 496
Single Nucleotide Polymorphisms (SNP) analysis ........................................................... 496
Short tandem repeat (STR) ........................................................................................ 497
Applications................................................................................................................... 497
Applications in criminology ......................................................................................... 498
Identification of remains of military personnel .............................................................. 499
Identification of remains of victims of mass disasters .................................................... 499
Parentage testing ...................................................................................................... 501
Gender determination ................................................................................................ 502
Companies developing molecular diagnostics for forensic science ......................................... 502
Molecular detection of biological warfare agents ............................................................. 503
Introduction to biological warfare agents........................................................................... 503
Role of PCR in the diagnosis of biological warfare agents ..................................................... 504
Multiplex PCR microarray assay to detect bioterror pathogens in blood ............................ 505
Laboratory diagnosis of Anthrax ....................................................................................... 505
Challenges in diagnosis of biological warfare agents ........................................................... 506
US government efforts for detection of biological warfare agents ......................................... 506
The US Army Medical Research Institute of Infectious Diseases....................................... 507
Homeland Security Advance Research Projects Agency .................................................. 507
Handheld Isothermal Silver Standard Sensor ................................................................ 508
Commercial development of diagnostic devices for biological agents ..................................... 508
Companies developing diagnostic devices for biological agents........................................ 508

- 18 -

Biodefence microarray ............................................................................................... 511

Identification of genetic markers of individual pathogens ............................................... 511
Microbial Identification System based on OptiChip ...................................................... 512
Hand-Held Advanced Nucleic Acid Analyzer .................................................................. 512
Nanogen's portable detection device ............................................................................ 512
Nanode Array Sensor Microchips ................................................................................. 512
MicroChemLab .......................................................................................................... 513
BioThreat Alert Test Strip ........................................................................................... 513
Benchtop living cell biosensor ..................................................................................... 513
BioForce NanoArray sensor technology ........................................................................ 514
QTL handheld biosensor ............................................................................................. 514
Analyte 2000 biosensor.............................................................................................. 514
Airborne bacterial spore detection technology ............................................................... 515
Destruction and detection of anthrax by lysin ............................................................... 515
Biosensor based on mass spectrometry of microorganisms's RNA .................................... 515
Bead ARray Counter .................................................................................................. 515
ProteinChip-based detection of bioterroism agents ........................................................ 516
TIGER biosensor ....................................................................................................... 516
The PathAlert Detection System ............................................................................... 516
VereThreat ............................................................................................................ 517
Concluding remarks about biodefense applications of diagnostics ......................................... 517

11. References ........................................................................................ 518

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1-1: Landmarks in development of molecular technology and its application to diagnosis ........... 26
1-2: Applications of molecular diagnostics ............................................................................ 47
2-1: Companies with products for nucleic acid isolation .......................................................... 57
2-2: Some commercially available real-time PCR systems ...................................................... 64
2-3: Applications of real-time PCR ....................................................................................... 66
2-4: A selection of companies with commercially available FISH diagnostics ............................. 92
2-5: Selected companies with RNA diagnostic tests ............................................................... 93
2-6: Companies involved in whole genome amplification ...................................................... 103
2-7: Companies involved in sequencing ............................................................................. 106
2-8: Companies involved in application of sequencing in molecular diagnostics ....................... 109
2-9: Comparison of methods of identification of unknown DNA sequences .............................. 110
2-10: Classification of methods of gene expression analysis ................................................. 111
2-11: A selection of companies with gene expression technologies ........................................ 117
2-12: Companies involved in developing PNA diagnostics ..................................................... 120
2-13: Companies with bead-based diagnostic assay platforms .............................................. 124
2-14: Companies developing nucleic acid lateral flow molecular diagnostics ............................ 126
3-1: Applications of biochip technology in relation to molecular diagnostics ............................ 128
3-2: Companies developing whole genome chips/microarrays ............................................... 134
3-3: Companies involved in biochips for molecular diagnostics .............................................. 135
3-4: Companies developing microfluidic technologies ........................................................... 140
3-5: Biosensor technologies with potential applications in molecular diagnostics...................... 141
3-6: Important applications of biosensors ........................................................................... 151
3-7: Companies involved in application of biosensors in molecular diagnostics ........................ 151
3-8: Selected labels for nucleic acid detection ..................................................................... 153
3-9: Selected companies with fluorescence and chemiluminescence products ......................... 154
3-10: Companies involved in molecular beacon manufacture and research ............................. 156
3-11: Selected companies involved in molecular imaging ..................................................... 165
3-12: Nanotechnologies with potential applications in molecular diagnostics ........................... 166
3-13: Companies developing nanomolecular diagnostics ...................................................... 175
4-1: Applications of protein biochips/microarrays ................................................................ 186
4-2: Companies involved in developing diagnostic applications of protein biochips ................... 193
4-3: Disease-specific proteins in the cerebrospinal fluid of patients ........................................ 196
5-1: Mutation detection technologies ................................................................................. 204
5-2: Technologies for SNP analysis .................................................................................... 212
5-3: A sampling of companies involved in technologies for SNP genotyping ............................ 221
5-4: Application of preimplantation genetic diagnosis in monogenic disorders ......................... 235
5-5: Companies involved in prenatal/preimplantation diagnostics .......................................... 236
5-6: CFTR genotyping in cystic fibrosis companies and technologies .................................... 238
5-7: X-linked immunodeficiency disorders .......................................................................... 242
5-8: Available molecular diagnostics for neurogenetic diseases ............................................. 249
5-9: Companies offering genetic screening tests directly to consumers .................................. 259
6-1: Genes that cause cardiovascular diseases.................................................................... 262
6-2: Molecular diagnostics for cardiovascular diseases: commercial development .................... 269
7-1: Molecular techniques for the diagnosis of infections ...................................................... 270
7-2: Commercially available molecular diagnostics for sepsis ................................................ 279
7-3: Bacteria that can be detected by molecular diagnostic tests ........................................... 287

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7-4: Commercially available molecular diagnostics for fungal infections ................................. 299
7-5: Viruses that can be detected by recombinant DNA methods .............................................. 302
7-6: Companies with molecular diagnostics for avian influenza virus H5N1 ............................. 326
7-7: Companies with molecular diagnostics for influenza virus H1N1 ..................................... 330
7-8: Commercially available molecular diagnostics for C. difficile ........................................... 340
7-9: Companies developing POC tests for the diagnosis of infections ..................................... 344
7-10: Selected companies involved in molecular diagnosis of infections ................................. 347
8-1: Estimated new cases of cancer in the US of most involved organs - 2011 ........................ 350
8-2: Tumor suppressor genes, their chromosomal location, function, and associated tumors .... 352
8-3: Viruses linked to human cancer ................................................................................... 354
8-4: A classification of molecular diagnostic methods in cancer ............................................. 357
8-5: Desirable characteristics of biomarkers for cancer ........................................................ 359
8-6: Approved monoclonal antibodies for cancer diagnosis ................................................... 369
8-7: Methods for comparison of gene-expression profilling in tumor specimens ....................... 370
8-8: Important cancer tests based on gene signatures ......................................................... 372
8-9: Impact of in vivo molecular imaging of cancer on oncology practice ................................ 386
8-10: Molecular diagnostic tests for breast cancer ............................................................... 391
8-11: Companies developing cancer molecular diagnostics ................................................... 433
9-1: Applications of molecular diagnostics in the biopharmaceutical industry .......................... 438
9-2: Molecular diagnostic technologies for drug discovery .................................................... 439
9-3: Molecular diagnostic technologies used for pharmacogenetic studies ............................... 439
9-4: Companies with novel molecular toxicology technologies .................................................. 440
9-5: Applications of molecular diagnostics in gene therapy ................................................... 445
9-6: Companies involved in transplant molecular diagnostics ................................................ 451
9-7: Companies involved in molecular diagnostics of blood transfusions ................................. 455
9-8: Pathogenic bacteria in food and targets for molecular diagnostic probes .......................... 467
9-9: Companies involved in molecular diagnostics for food-borne infections............................ 471
9-10: Testing for harmful prions in brain tissue from dead cattle ........................................... 473
9-11: Companies involved in developing molecular diagnostics for TSEs................................. 476
9-12: Companies involved in detection of genetically modified food ....................................... 477
9-13: Companies involved in companion diagnostics ............................................................ 484
9-14: Applications of point-of-care diagnosis ...................................................................... 485
9-15: Companies developing point-of-care diagnostic tests .................................................. 490
10-1: Forensic and legal applications of molecular diagnostics. ............................................. 497
10-2: Molecular technologies used for forensic applications .................................................. 502
10-3: Biological and chemical agents used as weapons of mass destruction ............................ 504
10-4: Biological warfare agents that can be identified by PCR methods .................................. 504
10-5: Companies developing detection devices for biological warfare agents .......................... 508

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Relation of molecular diagnostics to other technologies .................................................. 48

Rolling circle amplification technology .......................................................................... 75
A schematic view of the Invader operating system ........................................................ 83
Principle of fluorescent in situ hybridization .................................................................. 87
Repli-G system of QIAGEN ....................................................................................... 104
DNA sequencing process .......................................................................................... 106
Electrochemical detection of DNA .............................................................................. 123
Elements of a Scorpions primer................................................................................. 125
Affymetrix GeneChip technology ............................................................................... 129
Basic principle of a biosensor .................................................................................... 141
Surface plasmon resonance (SPR) technology ............................................................. 146
Use of DNA chips in diagnosing microbial infections ..................................................... 274
High throughput DNA pyrosequencing for pathogen discovery....................................... 278

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