Table Of ContentMolecular Diagnostics
Third Edition
Editor-in-Chief
George P. Patrinos
Associate Editors
Wilhelm J. Ansorge
Phillip B. Danielson
AMSTERDAM lBOSTON lHEIDELBERGlLONDON lNEWYORK lOXFORD lPARIS
SANDIEGO lSANFRANCISCO lSINGAPORE lSYDNEY lTOKYO
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List of Contributors
B.R. Ali United Arab Emirates University, Al-Ain, United L.R. Ferguson Nutrigenomics New Zealand, Auckland,
Arab Emirates New Zealand; The University of Auckland, Auckland,
New Zealand
W.J. Ansorge Ecole Polytechnique Federal Lausanne,
EPFL, Lausanne, Switzerland D.A. Forero Universidad Antonio Nariño, Bogotá,
Colombia
D.S. Atherton University of Alabama at Birmingham,
Birmingham, AL, United States J. Fredenburgh Scigen Scientific Inc., Gardena, CA,
United States
M.P.G. Barnett AgResearch Limited, Palmerston North,
New Zealand; Nutrigenomics New Zealand, Auckland, B.K. Gale University of Utah, Salt Lake City, UT,
New Zealand United States
W.C. Bell University of Alabama at Birmingham, J. Göransson Uppsala University, Uppsala, Sweden
Birmingham, AL, United States
W.E. Grizzle University of Alabama at Birmingham,
S.R. Brand Dana-Farber Cancer Institute, Boston, MA, Birmingham, AL, United States
United States
I. Grundberg Uppsala University, Uppsala, Sweden
G. Cane Uppsala University, Uppsala, Sweden
I.G. Gut Barcelona Institute for Science and Technology,
W. Chantratita Mahidol University, Bangkok, Thailand Barcelona, Spain
T. Conze Uppsala University, Uppsala, Sweden D.HeidemanVUUniversityMedicalCenter,Amsterdam,
The Netherlands
D. Corach Universidad de Buenos Aires, Buenos Aires,
Argentina I. Helbing Uppsala University, Uppsala, Sweden
F. Coun KU Leuven, Leuven, Belgium S. Henriksson Uppsala University, Uppsala, Sweden
P.B.DanielsonUniversityofDenver,Denver,CO,United I.Hernández-Neuta Stockholm University, Solna, Sweden
States; Center for Forensic Science Research and
F. Innocenti University of North Carolina at Chapel Hill,
Education, Willow Grove, PA, United States
Chapel Hill, NC, United States
J.T. den Dunnen Leiden University Medical Center,
M. Isaksson Uppsala University, Uppsala, Sweden
Leiden, Netherlands
M. Jarvius Uppsala University, Uppsala, Sweden
E. Dequeker University of Leuven, Leuven, Belgium
H. Jayamohan University of Utah, Salt Lake City, UT,
M.De Rycke Universitair Ziekenhuis& Vrije Universiteit
United States
Brussel (VUB), Brussels, Belgium
M. Kamali-Moghaddam Uppsala University, Uppsala,
A. Deshpande Los Alamos National Laboratory, Los
Sweden
Alamos, NM, United States
K. Kampourakis University of Geneva, Geneva,
S. Drmanac Complete Genomics, Inc., Mountain
Switzerland
View, CA, United States; BGI-Shenzhen, Shenzhen,
T. Katsila Universityof Patras School of Health Sciences,
China
Patras, Greece
R. Drmanac Complete Genomics, Inc., Mountain View,
B. Koos Uppsala University, Uppsala, Sweden
CA, United States; BGI-Shenzhen, Shenzhen, China
L. Koumakis Foundation for Research and Technology e
T. Ebai Uppsala University, Uppsala, Sweden
Hellas (FORTH), Heraklion, Greece
J.S.FarrarVirginia CommonwealthUniversity School of
P. Laissue Del Rosario University, Bogotá, Colombia
Medicine, Richmond, VA, United States
xv
xvi List of Contributors
U. Landegren Uppsala University, Uppsala, Sweden G. Potamias Foundation for Research and Technology e
Hellas (FORTH), Heraklion, Greece
C. Larsson Uppsala University, Uppsala, Sweden
N.ReisdorphUniversityofColoradoDenver,Aurora,CO,
K.M. Legg Center for Forensic Science Research and
United States
Education, Willow Grove, PA, United States
V. Romanov University of Utah, Salt Lake City, UT,
K.-J. Leuchowius Uppsala University, Uppsala, Sweden
United States
H.LiUniversityofUtah,SaltLakeCity,UT,UnitedStates
R. Samuel University of Utah, Salt Lake City, UT,
J.S. Liu Complete Genomics, Inc., Mountain View, CA,
United States
United States
K.C. Sexton University of Alabama at Birmingham,
A. Llerena Extremadura University Hospital and Medical
Birmingham, AL, United States
School, Badajoz, Spain
A. Sgourou Hellenic Open University, Patras, Greece
C.Lopez-CorreaGenomeQuebec,Montreal,QC,Canada
D. Sie VU University Medical Center, Amsterdam, The
C. Mathieu KU Leuven, Leuven, Belgium
Netherlands
H.E. McKiernan Center for Forensic Science Research
E.SistermansVUUniversityMedicalCenter,Amsterdam,
and Education, Willow Grove, PA, United States
The Netherlands
E. Mendrinou University of Patras School of Health
O. Söderberg Uppsala University, Uppsala, Sweden
Sciences, Patras, Greece
J.SonUniversityofUtah,SaltLakeCity,UT,UnitedStates
A. Mezger Stockholm University, Solna, Sweden
A. Squassina University of Cagliari, Cagliari, Italy
K. Mitropoulos The Golden Helix Foundation, London,
C. Staessen Universitair Ziekenhuis & Vrije Universiteit
United Kingdom
Brussel (VUB), Brussels, Belgium
C. Mizzi University of Malta, Msida, Malta
J. Stenberg Uppsala University, Uppsala, Sweden
L. Moens Uppsala University, Uppsala, Sweden
P.E.M. Taschner University of Applied Sciences Leiden,
Z. Mohamed University of Malaya, Kuala Lumpur,
Leiden, Netherlands
Malaysia
H. Tönnies Robert Koch-Institute, Berlin, Germany
J. Nelson University of Utah, Salt Lake City, UT,
J. Tost CEAeInstitut de Génomique, Evry, France
United States
G. Tzimas Technological Educational Institute of Western
M.NilssonStockholmUniversity,Solna,Sweden;Uppsala
Greece, Patras, Greece
University, Uppsala, Sweden
V. Velissariou Bioiatriki Health Services, Athens, Greece
L. Overbergh KU Leuven, Leuven, Belgium
E.ViennasUniversityofPatrasSchoolofHealthSciences,
A. Papachatzopoulou University of Patras School of
Patras, Greece
Health Sciences, Patras, Greece
S. Vig KU Leuven, Leuven, Belgium
K. Pardali Uppsala University, Uppsala, Sweden
P.S.WhiteLosAlamosNationalLaboratory,LosAlamos,
A.F. Patenaude Dana-Farber Cancer Institute, Boston,
NM, United States
MA, United States
C.T.WittwerUniversityofUtahSchoolofMedicine,Salt
G.P. Patrinos University of Patras School of Health
Lake City, UT, United States
Sciences, Patras, Greece; Erasmus University Medical
Center, Rotterdam, The Netherlands; United Arab A.WonkamUniversityofCapeTown,CapeTown,South
Emirates University, Al-Ain, United Arab Emirates Africa
P.C. Patsalis The Cyprus Institute of Neurology and X. Xun Complete Genomics, Inc., Mountain View, CA,
Genetics, Nicosia, Cyprus United States; BGI-Shenzhen, Shenzhen, China
B.A. Peters Complete Genomics, Inc., Mountain View, B. Ylstra VU University Medical Center, Amsterdam,
CA, United States; BGI-Shenzhen, Shenzhen, China The Netherlands
Preface, Third Edition
We are delighted to deliver to the scientific community the third edition of Molecular Diagnostics, perhaps the most
successful textbook in the field of molecular genetic testing, almost 12years from its first appearance in the scientific
literature.
In 2003, just 2years after the publication of the first draft of the Human Genome sequence, we proposed to edit a
textbook exclusively devoted to the description of molecular techniques used to identify the underlying genetic hetero-
geneityleadingtoinheriteddisorders.ThefirsteditionofMolecularDiagnosticswaspublishedinApril2005,followedby
the second edition in October 2009. This textbook has been available to the scientific community for over 10years now,
anditisclearlyconsideredtobethekeyreferenceinthefield,judgingfromthefollowing:(1)thelargenumberofcopies
soldworldwide;(2)thevariouspostgraduateandspecialisttrainingcoursesonMolecularDiagnosticsthathavebeenused
as syllabus and course material; (3) the adoption from universities as the textbook for related undergraduate courses and
curricula, which also led to its translation in 2008; and (4) the various positive reviews obtained not only from external
reviewers in scientific journals and elsewhere but also from fellow academics and students.
This has prompted Elsevier/Academic Press to request the compilation of a third edition, justified not only from
technological advances, particularly in high-throughput methods, but also from the intellectual revolution in biomedical
sciences.Inthisthirdeditionwedecidedtokeeptheoriginalstructureoftheprevioustwoeditions,sincethiswasoneofits
main innovative aspects, but we opted to expand the editorial team. We also decided to reshuffle the table of contents
completely, providing a succinct outline and a historical perspective of the low-throughput methods that set a solid basis
for the recent discoveries of the high-throughput methods together with a detailed overview of modern high-throughput
methodologies, such as next-generation sequencing and microarray-based methods, along with examples of their appli-
cations in a modern molecular genetic testing laboratory.
The contents of this book are divided into three parts. The first part is dedicated to the battery of the most modern
molecularbiologytechniquesandahistoricalperspectiveofthelow-throughputmethods.Inordertokeeppacewithrecent
developments, the majority of the chapters from the previous editions have been either merged into a few chapters or
omitted altogether, while being mentioned and fully referenced in the updated chapters. A large number of chapters
pertaining to high-throughput molecular diagnostic approaches, for example, microarrays, next-generation sequencing,
massspectrometry, next-generationsequencingcytogenomics,etc., havebeenincluded.The remainingchaptersfromthe
previouseditionshavebeencomprehensivelyupdatedtoincludenotonlytechnologyinnovationsbutalsonoveldiagnostic
applications. This resulted in the book being completely revamped with over half of its content being new compilations.
The second part attempts to integrate previously analyzed technology with different aspects of molecular diagnostics,
such as pharmacogenomics, molecular forensics and victim identification in mass disasters, and preimplantation genetic
diagnosis,whilenewemergingdisciplines,suchasnutrigenomics,genomeinformatics,andgenomicdatabases,havebeen
included. Finally, various everyday issues in a diagnostic laboratory, from genetic counseling to related ethical and
psychological issues to safety and quality management, are discussed in the third and final part of the book. As with the
previoustwoeditions,wefeelthattheinclusionofthelatterissuesinthisreferencebookhasgreatrelevancetooursociety.
Aswiththeprevioustwoeditions,ourefforthasbeenassistedbymanyinternationallyrenownedexpertsintheirfields
fromfivecontinentswhokindlyacceptedourinvitationtocompilethe29chaptersofthisbookandsharewithusandour
readerstheirexpertise,experience,andresults.Inaddition,wemadeanefforttoformulatethebookcontentssuchthatthe
notions described are explained in a simple language and terminology for the book to be useful not only to experienced
physicians, healthcare specialists, and academics but also to undergraduate medical and life science students. The
numerous self-explanatory illustrations and glossary clearly contribute to this end. Last, but not least, we provided the
means to resolve the previously reported deficiencies in variant nomenclature by including a chapter on the official gene
and genetic variation nomenclature at the very beginning of the textbook.
xvii
xviii Preface, Third Edition
We are grateful to those colleagues who provided constructive comments and criticisms on the previous two editions
andidentifieddeficienciesthathavebeen,hopefully,rectifiedinthisthirdedition.However,weexpectthatsomepointsin
thisbookcanstillbefurtherimproved.Thereforewewouldagainwelcomecommentsandcriticismfromattentivereaders,
whichwillcontributetoimprovingthecontentsofthisbookevenfurtherinitsfutureeditions.Wearealsogratefultothe
editors, Drs. Tari Broderick, Jeffrey Rossetti, and Tracy Tufaga at Elsevier, who helped us in close collaboration to
overcome encountered difficulties. We also express our gratitude to all of the contributors for delivering outstanding
compilationsthatsummarizetheirexperienceandmanyyearsofhardworkintheirfieldsofresearch.Weareindebtedto
GregHarris,whowasresponsibleforthedesignandthecoverofthisbook,andtotheproductionprojectmanagersKirsty
HaltermanandKarenEast,whohaverefinedthefinalmanuscriptpriortogoingintoproduction.Weoweourthankstothe
academic reviewers for their constructive criticisms on the chapters and their positive evaluation of our proposal for this
third edition.
Last,butnotleast,wewishtocordiallythankourfamilies,fromwhomwehavetakenaconsiderableamountoftimeto
devote to this project, for their patience and continuous support over the years.
Editor-in-Chief
George P. Patrinos
Department of Pharmacy, Faculty of Health Sciences
University of Patras, Patras, Greece
Associate Editors
Wilhelm J. Ansorge
École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
Phillip B. Danielson
Department of Biological Sciences, University of Denver
Denver, CO, United States of America
December 2016
Chapter 1
Molecular Diagnostics: Past, Present,
and Future
G.P. Patrinos1, P.B. Danielson2,3 and W.J. Ansorge4
1UniversityofPatrasSchoolofHealthSciences,Patras,Greece;2UniversityofDenver,Denver,CO,UnitedStates;3CenterforForensicScience
ResearchandEducation,WillowGrove,PA,UnitedStates;4EcolePolytechniqueFederalLausanne,EPFL,Lausanne,Switzerland
1.1 INTRODUCTION recurrentepisodesofacutepainduetovesselocclusion.In
principle, their findings set the foundations of molecular
Molecular (or nucleic acid-based) diagnosis of human
diagnostics, although the big revolution occurred many
disordersisreferredtoasthedetectionofgenomicvariants
years later.Atthat time, when molecular biologywasonly
that are pathogenic and/or benign in DNA and/or RNA
hecticallyexpanding,theprovisionofmoleculardiagnostic
samples in order to facilitate detection, diagnosis, sub-
services was inconceivable and technically not feasible.
classification, prognosis, and monitoring response to ther- The first seeds of molecular diagnostics were provided in
apy. Molecular diagnostics combines laboratory medicine
theearlydaysofrecombinantDNAtechnology,withmany
with the knowledge and technology of molecular genetics
scientists from various disciplines working in concert.
and has been enormously revolutionized over the last de-
cDNAcloningandsequencingwereatthattimeinvaluable
cades,benefitingfromdiscoveriesinthefieldsofmolecular
tools for providing basic knowledge on the primary
biology and genomic technologies (Table 1.1). The iden-
sequenceofvariousgenes.Thelatterprovidedanumberof
tification and fine characterization of the genetic basis of
DNA probes, allowing for analysis via southern blotting
inherited diseases is vital for the accurate provision of
of genomic regions, leading to the concept and application
diagnosis. Gene discovery, via high-throughput methods,
of restriction fragment length polymorphism (RFLP) to
such as next-generation sequencing or genome-wide asso-
track a variant allele from heterozygous parents to a high-
ciation studies, provides invaluable insights into the
risk pregnancy. In 1976, Kan and coworkers carried out,
mechanisms of disease, and genomic markers allow phy- forthefirsttime,prenataldiagnosisofa-thalassemia,using
sicianstonotonlyassessdiseasepredispositionbutalsoto hybridizationonDNAisolatedfromfetalfibroblasts.Also,
design and implement accurate diagnostic methods. The
Kan and Dozy (1978), implemented RFLP analysis to
latter is of great importance, as the plethora and variety of
pinpoint sickle cell alleles of African descent. This break-
moleculardefectsdemandstheuseofmultipleratherthana
through provided the means of establishing similar diag-
singlevariantdetectionplatform.Moleculardiagnosticshas
nostic approaches for the characterization of other genetic
gradually become a clinical reality with its roots deep into
diseases,suchasphenylketonurea(Wooetal.,1983),cystic
the basic science of gene expression and gene function. fibrosis (Farrall et al., 1986), and so on.
Atthattime,however,asignificanttechnicalbottleneck
had to be overcome. The identification of the pathogenic
1.2 HISTORY OF MOLECULAR
variant was possible only through the construction of a
DIAGNOSTICS: INVENTING THE WHEEL
genomicDNAlibraryfromtheaffectedindividual,inorder
In 1949, Pauling and his coworkers introduced the term to first clone the variant allele and then determine its
moleculardiseaseinthemedicalvocabulary,basedontheir nucleotide sequence. Again, many human globin gene
discovery that a single amino acid change at the b-globin mutations were among the first to be identified through
chain leads to sickle cell anemia, characterized mainly by such approaches (Busslinger et al., 1981; Treisman et al.,
1
MolecularDiagnostics.http://dx.doi.org/10.1016/B978-0-12-802971-8.00001-8
Copyright©2017ElsevierLtd.Allrightsreserved.
2 MolecularDiagnostics
TABLE1.1 Timelineofthe PrincipalDiscoveriesintheFieldofMolecularBiology,Which Influencedthe
DevelopmentofMolecularDiagnostics
Date Discovery
1949 Characterizationofsicklecellanemiaasamoleculardisease
1953 DiscoveryoftheDNAdoublehelix
1958 IsolationofDNApolymerases
1960 Firsthybridizationtechniques
1969 Insituhybridization
1970 Discoveryofrestrictionenzymesandreversetranscriptase
1975 Southernblotting
1977 DNAsequencing
1983 Firstsynthesisofoligonucleotides
1985 Restrictionfragmentlengthpolymorphismanalysis
1985 Inventionofthepolymerasechainreaction
1986 Developmentoffluorescentinsituhybridization
1988 DiscoveryofthethermostableDNApolymerasedoptimizationofthepolymerasechainreaction
1992 Conceptionofthereal-timepolymerasechainreaction
1993 Discoveryofstructure-specificendonucleasesforcleavageassays
1996 FirstapplicationofDNAmicroarrays
2001 Firstdraftversionsofthehumangenomesequence
2001 Applicationofproteinprofilinginhumandiseases
2002 LaunchoftheHapMapproject
2005 Introductionofhigh-throughputnext-generationsequencingtechnology
2008 Launchofthe1000GenomesProject
2013 IntroductionoftheCRISPRsystemforgeneediting
2014 Announcementofthesequencingofthehumangenomefor$1000
2015 LaunchofthePrecisionMedicineInitiativebyUSPresidentBarackObama
1983). In 1982, Orkin and his coworkers showed that a developed, setting the basis for variant screening and
number of sequence variations were linked to specific scanning methods. The first methods involved mismatch
pathogenic HBB gene variants. These groups of RFLPs, detection in DNA/DNA or RNA/DNA heteroduplexes
termed haplotypes (both intergenic and intragenic), have (Myers et al., 1985a,b) or differentiation of mismatched
provided a first-screening approach in order to detect a DNA heteroduplexes using gel electrophoresis, according
disease-causing variant. Although this approach enabled to their melting profile (Myers et al., 1987). Using this
researchers to predict which HBB allele was pathogenic, laborious and time-consuming approach, a number of
significantly facilitating mutation screening, no one was in variant sequence alleles have been identified, which made
the position to determine the exact nature of the disease- possible the design of short synthetic oligonucleotides that
causing mutation, as many different HBB gene variants were used as allele-specific probes onto genomic Southern
werelinkedtoaspecifichaplotypeindifferentpopulations blots. This experimental design was quickly implemented
(furtherinformationisavailableathttp://globin.bx.psu.edu/ for the detection of b-thalassemia mutations (Orkin et al.,
hbvar; Patrinos et al., 2004; Giardine et al., 2014). 1983; Pirastu et al., 1983).
Atthesametime,inordertoprovideashortcuttoDNA Despite intense efforts from different laboratories
sequencing, a number of exploratory methods for pin- worldwide,thediagnosisofinheriteddiseasesontheDNA
pointing pathogenic variants in patients’ DNA were level was still underdeveloped and therefore still not ready
MolecularDiagnostics:Past,Present,andFuture Chapter | 1 3
to be implemented in clinical laboratories for routine activity of resolvase enzymes T4 endonuclease VII
analysisofpatientsduetothecomplexities,costs,andtime and T7 endonuclease I to digest heteroduplex DNA
requirementsofthetechnologyavailable.Itwasonlyaftera formed by annealing wild type and mutant DNA
few years that molecular diagnosis entered its golden era (Mashal et al., 1995). Digestion fragments indicate
withthediscoveryofthemostpowerfulmolecular biology the presence and the position of any variants. A vari-
tool since cloning and sequencing, the polymerase chain ationofthe themeinvolvesthe use of chemical agents
reaction (PCR). for the same purpose (Saleeba et al., 1992). Another
enzymatic approach for variant detection is the oligo-
nucleotide ligation assay (Landegren et al., 1988;
1.3 THE POST-POLYMERASE CHAIN
Chapter 3); ligation was also one of the main princi-
REACTION REVOLUTION
ples of one of the most widely used next-generation
The discovery of PCR (Saiki et al., 1985; Mullis and sequencing approaches (see also Chapter 8).
Faloona, 1987) and its quick optimization, using a ther- 2. Electrophoretic-based techniques. This category is
mostable Taq DNA polymerase from Thermus aquaticus characterized by a plethora of different approaches
(Saiki et al., 1988), has greatly facilitated and in principle designedforthescreeningofknownorunknownmuta-
revolutionized molecular diagnostics. The most powerful tions, based on the different electrophoretic mobility of
feature of PCR is the large amount of copies of the target the mutant alleles, under denaturing or nondenaturing
sequencegeneratedbyitsexponentialamplification,which conditions. Single-strand conformation polymorphism
allows the identification of a known mutation within a (SSCP) and heteroduplex analyses (HDA; Orita et al.,
single day, rather than months. Also, PCR has markedly 1989; see Chapter 3) were among the first methods
decreased or even diminished the use of radioactivity for designed to detect molecular defects in genomic loci.
routine molecular diagnosis. This has allowed molecular In combination with capillary electrophoresis, SSCP
diagnosticstoentertheclinicallaboratoryfortheprovision and HDA analysis now provide an excellent, simple,
of genetic services, such as carrier or population genetic andrapidvariantdetectionplatform withlowoperation
screening, prenatal diagnosis of inherited diseases, or, in costs and, most interestingly, the potential of easily be-
recent years, the identification of unknown variants, in ing automated, thus allowing for high-throughput anal-
closecollaborationwithresearchlaboratories.Thereforeby ysis of patients’ DNA. Similarly, denaturing gradient
being moved to their proper environment, the clinical lab- gel electrophoresis (DGGE) and temperature gradient
oratory, molecular diagnostics could provide the services gel electrophoresis can be used equally well for variant
for which they have been initially conceived. alleledetection(seeChapter3).Inthiscase,electropho-
The discovery of PCR also has provided the founda- retic mobility differences between a wild type and
tions for the design and development of many variant variantallelecan be “visualized” in a gradientof dena-
detection schemes, based on amplified DNA. In general, turing agents, such as urea and formamide, or of
PCRiseitherusedforthegenerationofDNAfragmentsto increasing temperature. A less common variant detec-
be analyzed or is part of the detection method. The first tiontechniqueistwo-dimensionalgenescanning,based
attempt was the use of restriction enzymes (Saiki et al., ontwo-dimensionalelectrophoreticseparationofampli-
1985) or oligonucleotide probes, immobilized onto mem- fied DNA fragments, according to their size and base
branes or in solution (Saiki et al., 1986), in order to detect pair sequence. The latter involves DGGE, following
the existing genetic variation, in particular the sickle cell the size separation step.
disease-causing mutation. In the following years, an even 3. Solid phase-based techniques. This set of techniques
larger number of variant detection approaches have been consists of the basis for most of the present-day muta-
developed and implemented (see also Chapter 3). These tion detection technologies, since they have the extra
techniques can be divided roughly into three categories, advantage of being easily automated and hence are
depending on the basis for discriminating the allelic highly recommended for high-throughput mutation
variants: detection orscreening.Afast,accurate,andconvenient
methodforthedetectionofknownmutations isreverse
1. Enzymatic-based methods. RFLP analysis was histori-
dot-blot, initially developed by Saiki et al. (1989) and
cally the first widely used approach, exploiting the al- implemented for the detection of HBB gene variants
terations in restriction enzyme sites, leading to the leading to b-thalassemia. The essence of this method
gain or loss of restriction events (Saiki et al., 1985).
is the utilization of oligonucleotides, bound to a mem-
Subsequently, a number of enzymatic approaches for brane, as hybridization targets for amplified DNA.
variant allele detection have been conceived, based Some ofthistechnique’sadvantagesarethatonemem-
onthedependenceofa secondary structure on thepri-
brane strip can be used to detect many different known
mary DNA sequence. These methods exploit the
mutationsinasingleindividual(aonestrip-one patient
4 MolecularDiagnostics
typeofassay),thepotentialofautomation,andtheease disciplines to intensify their research efforts to improve by
of interpretation of the results, using a classical avidin- orders of magnitude the existing methods for genomic
biotin system. However, this technique cannot be used variantdetection,tomakeavailabledatasetswithgenomic
for the detection of unknown mutations. Continuous variationandanalyzethesesetsusingspecializedsoftware,
development has given rise to allele-specific hybridiza- to standardize and commercialize genetic tests for routine
tion of amplified DNA [PCR-ASO (Allele Specific diagnosis, and to improve the existing technology in order
Oligonucleotide), Chapter 3] on filters, recently to provide state-of-the-art automated devices for high-
extended to DNA oligonucleotide microarrays (see throughput genetic analysis.
Chapter 18) for high-throughput mutation analysis The biggest challenge, following the publication of the
(Gemignani et al., 2002; Cremonesi et al., 2007). In humangenomedraftsequence,wastoimprovetheexisting
particular, oligonucleotides of known sequence are variant detection technologies to achieve robust, cost-
immobilized onto appropriate surfaces, and hybridiza- effective, rapid, and high-throughput analysis of genomic
tion of the targets to the microarray is detected, mostly variation. Also, the increased pace of novel variant detec-
using fluorescent dyes. tionandgenediscoverydictatedtheharmonizationofgene
nomenclature,aneffortthatwasspearheadedbytheHuman
Thechoiceofthevariantdetectionmethodisdependent
Genome Variation Society (http://www.hgvs.org; see also
upon a number of variables, including the variation spec-
Chapter2).Since 2005,genomictechnologyhasimproved
trum of a given inherited disorder, the available infra-
rapidly, and new high-throughput variant detection tech-
structure, the number of tests performed in the diagnostic
niques have become available, whereas old methodologies
laboratory, and issues of intellectual properties (see also
have evolved to fit into the increasing demand for auto-
Section 1.5.1). Most of the clinical diagnostic laboratories
mated and high-throughput screening or were gradually
have not invested in expensive high-technology in-
abandoned.Thechromatographicdetectionofpolymorphic
frastructures, since the test volumes (the number of tests)
changes of pathogenic variants using denaturing high-
expected to be performed have not been large enough to
performance liquid chromatography (DHPLC; for review,
justify the capital investment. Therefore simple “home-
seeXiaoandOefner,2001)isoneofthenewtechnologies
brew”screening tests such as SSCP and HDA were and
that emerged. DHPLC reveals the presence of a genetic
still are the methods of choice for many clinical labora-
variation by the differential retention of homo- and het-
tories,astheyallowforrapidandsimultaneousdetectionof
eroduplex DNA on reversed phase chromatography under
different sequence variations at a detection rate of close to
partial denaturation. Single-base substitutions, deletions,
100%. Although DNA amplification has significantly
andinsertionscanbedetectedsuccessfullybyultravioletor
facilitated the expansion of molecular diagnostics, it
fluorescence monitoring within 2 to 3min in unpurified
nonetheless has a number of limitations, such as amplifi-
PCR products as large as 1.5-kilo bases. These features,
cation of cytidine-guanine repeat-rich regions, the error-
prone features of Taq polymerase (at a range of 10(cid:1)4 to together with its low cost, make DHPLC one of the most
10(cid:1)5 per nucleotide), and so on. Finally, it is noteworthy powerful tools for mutational analysis. Also, pyrose-
quencing,anongel-basedgenotypingtechnology,provides
that despite the wealth of variant detection methodologies,
a very reliable method and an attractive alternative to
DNA sequencing, particularly in the era of whole genome
DHPLC. Pyrosequencing detects de novo incorporation of
sequencing and the breakthroughs in next-generation
nucleotides based on the specific template. The incorpora-
sequencing technology, is considered the golden standard
tion process releases a pyrophosphate, which is converted
and the definitive experimental procedure for variant call-
to ATP and followed by luciferase stimulation. The light
ing. However, the costs for the initial investment and the
produced, detected by a charge couple device camera, is
difficulties for standardization and interpretation of
“translated” to a pyrogram, from which the nucleotide
ambiguousresultshaverestricteditsuseespeciallytobasic
sequence can be deducted (Ronaghi et al., 1998). This
research laboratories.
approach constituted the basis for the development of the
first next-generation sequencing approaches by 454 Life
1.4 MOLECULAR DIAGNOSTICS IN THE
Technologies (see also Chapter 8).
POST-GENOMIC ERA
One of the major advances was the invention of the
In February 2001, with the announcement of the first draft real-time PCR and the numerous variations of this theme
sequence of the human genome (International Human (Holland et al., 1991; see Chapter 4). The method allows
Genome Sequencing Consortium, 2001; Venter et al., for the direct detection of the PCR product during the
2001) and subsequently with the genomic sequence of exponential (mid-log) phase of the reaction, therefore
other organisms, molecular biology has entered into a new combining amplification and detection in one single step.
erawithunprecedentedopportunitiesandchallenges.These The increased speed of real-time PCR is due largely to
tremendous developments put pressure on a variety of reduced cycles, the removal of post-PCR detection
