Detection of Non-Amplified Genomic DNA

319 Pages·2012·7.11 MB·English

by  Alessia Finotti

Detection of Non-Amplified Genomic DNA
SOFT AND BIOLOGICAL MATTER
“Soft and Biological Matter” is a series of authoritative books covering
establishedandemergentareasintherealmofsoftmatterscience,including
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PeterSchall,UniversityofAmsterdam,theNetherlands
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Giuseppe Spoto • Roberto Corradini
Editors
Detection of Non-Amplified
Genomic DNA
123
Editors
Prof.GiuseppeSpoto Prof.RobertoCorradini
DepartmentofChemicalScience DepartmentofOrganic
UniversityofCatania andIndustrialChemistry
Catania UniversityofParma
Italy Parma
Italy
ISSN2213-1736 ISSN2213-1744(electronic)
ISBN978-94-007-1225-6 ISBN978-94-007-1226-3(eBook)
DOI10.1007/978-94-007-1226-3
SpringerDordrechtHeidelbergNewYorkLondon
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Preface
The extraordinaryeffortin the sequencingof living organismgenomes, including
the human genome project, is continuously providing new targets for the use of
geneticinformationasspecificmarkersforanalysisofsamplesofbiologicalorigin;
this process has prompted towards the developmentof new methodologiesfor an
even more simpler, faster and cheaper detection of nucleic acids to be used for
bothapplicativeandresearchneedsindifferentareas,includingclinicaldiagnosis,
environmentalmonitoringandfoodcontrol.
Most of the currently available methodologies to detect the DNA base se-
quences exploit the recognition capabilities of nucleic acids and are based on the
hybridization reaction between a probe oligonucleotide carrying a known base
sequenceandthecounterpartpresentintheunknownDNAtarget.Mostoftheabove
methodsrequiretheamplificationofthetargetspeciesasaconsequenceofthevery
smallamountofDNAmaterialoftenavailableanddetecttheduplexformationby
using indicators (labels) and transducers able to generate a detectable signal as a
consequenceofthespecifichybridizationevent.
Thepolymerasechainreaction(PCR)isthemostwidelyadoptedtechniqueused
to amplifya targetDNA. Itamplifiesa single piece of DNA across severalorders
of magnitude, duplicating millions or more copies of a particular DNA sequence,
inorderto detectthegeneticmaterialmoreeasily.Real-timePCR ispresentlythe
goldenstandardforquantitativeDNAanalysis.
ThePCRamplificationofthetargetsequencesisusedsinceitenhancesboththe
specificityandthesensitivityoftheassaybyincreasingthetargetconcentration.
Suchenhancementisbalancedbytheneedtousecomplexreactionmixturesand
bythe potentialcontaminationofthe geneticsample bothresultingin ambiguities
and errors in the final DNA detection process. Moreover, PCR amplification
methods suffer from drawbacks such as the difficulty in amplifying large target
DNA molecules (more than about 10000 bases) and the possible amplification of
DNAsequencesdifferentfromthetargetresultinginnon-specificsignals.
Theconceptofamplificationhasalsobeenextendedtoothertypesofschemes,
inparticularthoseworkingunderisothermalconditions.
v
vi Preface
Sequence-specificityisoneofthemajorissuesinmodernDNA-baseddiagnos-
tics. Single point mutations are very important markers of alterations linked to
genetic diseases and tumour insurgence. Furthermore, the identification of single
nucleotide polymorphisms(SNPs), variation of a single nucleotide in the genome
sequence,isofgreatimportanceintheclassificationofbiologicalmaterial,allowing
to discriminate between individuals, varieties and species. Identification of SNP
is one of the major outcome of the modern massive sequencing technologies.
Detection of SNP and point mutationsis not easily performedby PCR platforms,
andspecializedtechniquesarecontinuouslybeingdevelopedtoallowbettersingle-
baselevelrecognitionability.
DNA detection is expected to be significantly improved by using simple and
economic detection methods which require minimal DNA modifications and pro-
vide enhanced signal amplification. In this perspective, the direct analysis of
non-amplified genomic DNA is an excellent cost-effective alternative that can be
achieved by using ultrasensitive DNA detection protocols, since extra labour and
costsfromtheamplificationprocedurearereduced.
Scientificliteraturepublishedoverthelast10yearshasshownthatnon-amplified
DNAdetectioncanbeachievedbyadoptingdifferenttechniques.Suchtechniques
haveallowedthecommercializationofinnovativeplatformsforDNAdetectionthat
areexpectedtobreakintotheDNAdiagnosticmarketthatisexpectedtobevalued
at $20.1 billion in 2013 with an annual growth rate of 13.7% between 2008 and
2013.In particular,new diagnostic assays for the PCR-free detection of DNA are
expectedtointroduceanewsegmentthatwillcompetewiththe$9.5billionin2013
PCR-baseddiagnosticassaymarket.
The enhanced sensitivity required when non-amplified genomic DNA is going
to be detectedhaspromptedeffortsaimedat identifynew strategiesusefulforthe
ultrasensitiveDNA detection.Itisclear todaythatultrasensitivitycanbe obtained
bycombiningspecificmaterialswithspecificdetectiontools.
Advanced materials play different roles in the ultrasensitive detection. For
instance, nanostructures such as metallic nanoparticles have been widely inves-
tigated and the specific optical properties of gold nanoparticles have prompted
the developmentof new methods in DNA sensing exploiting the solutions colour
changegeneratedby a controlledaggregationof the colloidalnanoparticlesor the
specificdetectedopticalsignalenhancementgeneratedbymetallicsurfaceplasmon
interferences.Cationicconjugatedpolymershavebeendeeplyinvestigatedfortheir
abilitytoproduceamplifiedsignaluponinteractionwithdsDNA.
Probe selection, surface composition and architecture play a fundamental role
intheenhancedsensitivitydetectionofnon-amplifiedDNA.Someofthephysical
processeswhichcanbeusedtogenerateasignalinsensorsarestronglydependent
on the surface architecture and are greatly improved by the possibility to control
distances between the captured DNA analyte and the surface with nanometer
precision.Thisenablestodistinguishbetweenshort-andlong-rangeeffects,which
is particularlyimportantin the case of opticaldetection based on plasmonics. For
the generation of strong signals, the advantages offered by specifically designed
probes such as peptide nucleic acids (PNA), locked nucleic acids (LNA), or
Preface vii
morpholinooligonucleotides(MO)canbeexploited.Inparticular,syntheticprobes
offerimprovedselectivity and specificity in targetingcomplementarynucleic acid
sequences that can be tuned by properly designing their structure. Pushing the
analysisdowntoverylowdetectionlimitscanbeachievedonlyiftheprobe-DNA
interactionissufficientlystrongtopreventdissociationatverylowconcentrations;
thusenhancingtheperformancesof probesis oneoftheissues to becontrolledin
order to design new ultrasensitive methods. PNA and LNA are particularlysuited
forultrasensitivedetection,duetotheirhighDNAaffinity.Cationicpolythiophene
polymershasbeenusedfortheultrasensitivedetectionofnon-amplifiedDNAsince
their intrinsic fluorescence quenches as a result of the planar, highly conjugated
conformation adopted by the polymers when complexed with a single-strand
DNA probe but increases again after hybridization with the perfectly matched
complementarystrand.
Opticalandelectrochemicaldetectiontoolsareamongthemostwidelyinvesti-
gatedfortheanalysisofnon-amplifiednucleicacids. Boththemethodshavebeen
applied to the direct detection of genomic DNA and non-amplified RNAs from
bacteria. Optical detection of genomic DNA has gained a new impulse in recent
years after the advent of surface plasmon resonance (SPR). The SPR sensitivity
in the direct detection of unamplified genomic DNA has been evaluated by using
genomic DNA extracted from different living organisms. The limitation suffered
bySPRintheparalleldetectionofdifferentprobe/targetinteractionsareovercome
bySPRimagingwhiletheultrasensitivityisobtainedbyadoptingspecificmetallic
nanoparticlesignalenhancementprotocols.
Biosensors based on piezoelectric crystals have been also used to detect un-
amplified genomic DNA. The immobilization of oligonucleotide probes on the
piezoelectriccrystalsurfaceallowsthecombinationofthehighsensitivitytomass
changesofthepiezoelectrictransducerwiththehighspecificityofthehybridization
reaction between the probe and the nucleic acid targetand providesa tool for the
labelfreedetectionofunamplifiedDNA.
Finally, next generation sequencing technologies have been greatly improved,
thanks to the 1000$ genome challenge, and modern platforms are very close to
this goal. Thus redundant information on the genome of specific specimens is
also a possibility becomingmore and more feasible at the point of care level and
certainly the possibility to have the complete screen of genomic information at
the nucleotide sequence level is going not only to offer new services, but also to
substantiallychangesomeofoursocieties’fundamentalissues,suchashealthand
foodmanagement.
The brief description of the topic above makes it possible to understand just
how heterogeneous the interests and the professional background of the people
involvedare. In fact, chemists, biologists, biotechnologistsand physicistshave all
devotedtheirattentiontothefieldofnucleicaciddetectionusingtheirowncultural
background.Moreover,thewidespectrumofinformationprovidedhasestablished
a multi-disciplinaryapproachbut,at the same time, hasexpandedthe information
sourceswhicharetobeconsideredinthefield.
viii Preface
For all these reasons a book like this, where topics in the field of detection of
non-amplified DNA are reviewed, is the result of multi-disciplinary contributors.
The across-the-board relevance of the topics discussed in the book together with
the even greater scientific and economic importance of new diagnostic tools for
genomicanalysesaretwofundamentalreasonstoexpectahighlevelofinterestin
the readers. The interest shown in the subject by academic departments, colleges
andinstitutesinvolvedin thefield wouldappearto showthatthebookwouldalso
haveeducationalpotential.
The evolution of the topic discussed is itself a factor to be considered when
evaluatingtheinterestitmaygenerate.
The book starts with two chapter dedicated to the most important fields of
application of the DNA-detection technologies, that is health and food, which
account for the most important economic issues presumably enjoying the major
advantages from the availability of new simplified, fast and reliable methods for
massive detection of DNA sequences. Chapter 1 deals with the most important
issueswhicharepresentlyatstakeinmedicalsciences,fromnewbornsafetyarising
from pre-natal diagnostics to tumour progression evaluation and early diagnosis,
andlisttheexpectationsinthisfieldwhichcanbeforeseenforthenextyears.
Chapter2reportstheenormousprogressmadepossiblebyDNAanalysisinthe
fieldoffoodsciences,withapplicationtofoodsafety,traceabilityandauthenticity.
This field, being of huge social and economic relevance, offers the possibility to
applynewDNA detectionschemesto the developmentof newandunprecedented
services, which will be made easier to performby methodsdetecting unamplified
genomicDNAwithouttheuseofcomplexapparata.
Ofcoursethesedesideratacouldnotbesatisfiedifnewgenerationsofmaterials
and detection schemes would not have been developed by chemists and material
scientists. Thus, the next section deals with the materials and detection schemes
neededforobtainingultrasensitivediagnostictechniques,whichareapre-requisite
for unamplified DNA detection. Chapter 3 deals with the use of nanostructured
materialssuchasnanoparticles,nanowiresandnanogaps,whoseavailabilitypaved
the way for unamplified DNA detection. Chapter 4 deals with the molecular
probes needed to obtain high fidelity of the DNA capture in sensory systems,
and introduces the field of chemically engineered oligonucleotide analogs which
allow to reach higher levels of sensitivity and selectivity, such as locked nucleic
acids(LNA),morpholinooligonucleotides(MO)andpeptidenucleicacids(PNA),
together with an explanation of how these molecules can be linked to the sensor
and to other reporting groups for ultrasensitive detection. Molecular probes used
to directly visualize the genomic DNA by optical methods are also described.
Chapter5describescationicconjugatedpolymers(CCP)whichhavedemonstrated
great potential as DNA-sensing materials and how their optical properties can be
changed in the hybridization states. The application of these materials to direct
genomicDNAdetectionisalsodiscussed.
The technical advancements made in signal collection are also responsible for
the crossing of the threshold needed to directly detect unamplified DNA. Thus in
PartIII,a seriesof contributiondealingwith the analyticaltechniquesis reported.
Preface ix
In Chap. 6, the advances in DNA detection by optical techniques, including
colorimetric, fluorescence, luminescence, surface plasmon resonance (SPR) and
Raman scattering assays are reported, with up-to-date description of strategies
andtechnologiesforultrasensitiveandunamplifiedDNAanalysis.Electrochemical
techniques are another key point in modern DNA genosenors, and in Chap. 7
the most recent advances in this field are discussed, with a description of how
newfabricationtechniquesandnanomaterialshaveimprovedtheabilitytoprovide
rapid,multiplexedelectrochemicalDNAdetectionessentialforpointofcareclinical
diagnostics. The third large class of mechanical sensors is described in Chap. 8
dealing with piezoelectric sensing. In this chapter, the detection principle and the
approachesusedinDNA-basedsensingformicrosatelliteandtargetsequenceDNA
detection will be presented and discussed, together with a useful discussion on
sample treatment and probe chemistry. Surface plasmon resonance and related
techniques are among the most useful tools for label-free bioanalysis, and this
has allowed to more easily achieve PCR-free detection of DNA. In Chap. 9, the
principlesof thesetechniquesandtheirmostrecentapplicationsto genomicDNA
analysisarediscussed.
ModerntechnologiesandsurfacearchitecturesallowtoperformDNAdetection
inamultimodalapproach.Chapter10showsaninstructiveexampleofhowthese
elementscan be combinedfor the ultrasensitive detection of DNA samples, using
electrochemicalandsurfaceplasmonenhancedfluorescence.
FormostscientistsinterestedinDNAsensing,themostimportantissuesarethe
incredibleadvancesinsequencingtechnologies.Inthisfieldalso,non-amplificative
techniques have become more and more competitive. Chapter 11 gives excellent
and updated information about these next-generation sequencing techniques, and
guides the reader into this complicated field, which represents some of the most
advancedfrontiersofhumantechnologiessofarreached.
We are really gratefulto all the qualifiedscientists, who arelisted in thelist of
authors,fortheirpreciouscontributiontothisbook,bringingtheirexperiencesand
know-how and sharing these with our readers. We are also indebted to Dr. Maria
Bellantone,SeniorPublishingEditor,Springer,forherencouragement,supportand
patience.
Catania GiuseppeSpoto
Parma RobertoCorradini