DNAchips

1. DNAarrays
applications
fluorescence as an optical detection method fluorescenceasanopticaldetectionmethod
fabricationmethods
2. DNAseparation
conventional separation by gel electrophoresis conventionalseparationbygelelectrophoresis
electrophoresis
separationinmicroscaledevices
electroosmosis + electrophoresis electro osmosis electrophoresis
3. DNAsequencingandamplification
Sangermethodforsequencing
PCR amplifyquantitiesofDNAforsequencing C a p y qua t t es o o seque c g
microscalePCRchips
EECE432 2011 KCheung 1
Furtherreading
[1] B.Alberts,etal,"MolecularBiologyoftheCell,"
Th ti Th St t d F ti f DNA i il bl t ThesectiononTheStructureandFunctionofDNAisavailableat:
http://www.ncbi.nlm.nih.gov/books/NBK26821/#A597
DNAsequencing:
http://wwwncbi nlm nih gov/books/NBK26837/#A1583 http://www.ncbi.nlm.nih.gov/books/NBK26837/#A1583
Viewingmoleculesusingfluorescencemicroscopy:
http://www.ncbi.nlm.nih.gov/books/NBK26880/#A1737
[2] L.Bousse,C.Cohen,T.Nikiforov,A.Chow,A.R.KopfSill,R.Dubrow,andJ.W.Parce,
"Electrokinetically ControlledMicrofluidicAnalysisSystems,"AnnualReviewof
BiophysicsandBiomolecular Structure,vol.29,pp.155181,2000.
[3] C H M t l M A B d D T B k "Mi f b i t d d i f G ti [3] C.H.Mastrangelo,M.A.Burns,andD.T.Burke,"MicrofabricateddevicesforGenetic
diagnostics,"ProceedingsoftheIEEE,vol.86,pp.17691787,1998.
[4] M.U.Kopp,A.J.deMello,andA.Manz,"ChemicalAmplification:ContinuousFlowPCR
onaChip,"Science,vol.280,pp.10461048,1998. p pp
[5]MANZ,A.,EFFENHAUSER,C.S.,BURGGRAF,N.,HARRISON,D.J.,SEILER,K.&FLURI,K.
(1994)Electroosmoticpumpingandelectrophoreticseparationsforminiaturized
chemicalanalysissystems.JournalofMicromechanicsandMicroengineering,4,257.
EECE432 2011 KCheung 2
1.DNAarrays
Learning Objectives LearningObjectives
Attheendofthissectionthestudentshouldbeableto:
describe the basic structure of DNA and what is describethebasicstructureofDNAandwhatis
meantbycomplementarity
describeDNAarrays howtheyaremade
(Affymetrixchip)andwhataretheirapplications
describetheprincipleandpurposeofDNAprobe
hybridization assays hybridizationassays
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DNAbasics
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DNAbasics
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Nucleotides
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ComplementarybasepairsinDNA
The shapes and chemical structure of the bases allow hydrogen bonds to form
efficiently only between A and T and between G and C where atoms that are able to
EECE432 2011 KCheung
efficiently only between A and T and between G and C, where atoms that are able to
form hydrogen bonds.
Biological Physics, P. Nelson
7
DNAdoublehelix
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FromDNAtoProtein
Geneticinformation is
storedinDNAdoublehelix
Transcription
Translation
flowofinformationinacell
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ProteinSynthesis
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DNAarrays
DNAarraysareglasssurfacesbearingarraysofDNAfragmentsatdiscrete
addresses,atwhichthefragmentsareavailableforhybridization.
TheDNAspotsonthechiparehybridizedtoacomplexsampleof p p y p p
fluorescentlylabelledDNAorRNAinsolution.
ssDNAcanbechemicallysynthesizedupto120bases(oligonucleotides)
upto20b,complementarystrandattachonlyincaseofperfectmatching
DNA sample that contains a complementary sequence to a polynucleotide DNAsamplethatcontainsacomplementarysequencetoapolynucleotide
chainonaspotwillhybridize.
Hybridizationisusuallydetectedbyhavingafluorescentprobeattachedto
DNAsamples.
NonlabeledtechniquessuchasSPRorimpedancearealsopossiblefor
detection.
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ApplicationsofDNAarrays
1. QuantifymRNA transcribedfromdifferentgenesandwhichencode
differentproteins. RNAisextractedfrommanycells,ideallyfroma
singlecelltype,thenconvertedtocDNAorcRNA.
2. Identifygeneticvariationinindividualsandacrosspopulations.
Microarraysarealsobeingusedtoidentifygeneticvariationin
individuals and across populations. Short oligonucleotide arrays can be individualsandacrosspopulations.Shortoligonucleotidearrayscanbe
usedtoidentifythesinglenucleotidepolymorphisms(SNPs)thatare
thoughttoberesponsibleforgeneticvariationandthesourceof
susceptibilitytogeneticallycauseddiseases.Generallytermed
"genotyping" applications, chips may be used in this fashion for forensic genotyping applications,chipsmaybeusedinthisfashionforforensic
applications,rapidlydiscoveringormeasuringgeneticpredispositionto
disease,oridentifyingDNAbaseddrugcandidates.TheseSNP
microarraysarealsobeingusedtoprofilesomaticmutationsincancer.
Amplifications and deletions can also be detected using comparative Amplificationsanddeletionscanalsobedetectedusingcomparative
genomichybridizationinconjuctionwithmicroarrays.
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ApplicationsofDNAarrays
3. Expressionanalysis Expression
profiling to make comparisons bet een profiling,tomakecomparisons between
similarcelltypes.
Becausemanyproteinshaveunknownfunctions,
and because many genes are active all the time in andbecausemanygenesareactiveallthetimein
allkindsofcells,researchersusuallyuse
microarraystomakecomparisonsbetweensimilar
celltypes.
l l f b Forexample,anRNAsamplefrombraintumor
cells,mightbecomparedtoasamplefromhealthy
neuronsorglia.ReportersthatbindRNAinthe
tumor samplebutnotinthehealthyonemay p y y
indicategenesthatareuniquelyassociatedwith
thedisease.
Typicallyinsuchatest,thetwosamples'cDNAs are
t d ith t di ti t l bli
i h h d d
taggedwithtwodistinctcolors,enabling
comparisononasinglechip.Researchershopeto
findmoleculesthatcanbetargetedfortreatment
withdrugsamongthevariousproteinsencodedby
Sincetherearehundredsor
thousandsofdistinctreporterson
anarray,eachmicroarray
experimentcanaccomplishthe
equivalent of thousands of genetic
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diseaseassociatedgenes.
equivalentofthousandsofgenetic
testsinparallel.
13
TwotypesofDNAlibraries
genomicDNA: complementaryDNA(cDNA):
cleavetheentiregenomeofacelland
cloneeachfragment
canproduceaverylargenumberof
DNAfragmentsontheorderofa
onlythoseDNAsequencesthatare
transcribedintomRNAandthusare
presumedtocorrespondtoprotein
encodinggenes
millionforamammaliangenome
representarandomsampleofallof
theDNAsequencesinanorganism
extractthemRNA(orapurified
subfractionofthemRNA)fromcells
andthenmakeacomplementaryDNA
(cDNA)copyofeachmRNAmolecule
present present
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cDNAarrays
ThesearraysaremadebyroboticdepositionofDNAspotsthatare~50
150mindiameterontoacoatedglasssurface.Usually,PCRamplified
insertsfromcDNAclonesarearrayed.
OnecanhaveDNAchipsthatbear10,000spots,whichrepresentupto
10,000genesonanareaof3.6cm
2
.Probesrequire<600ngofmRNAper , g q g p
samplefora10,000spotDNAchip.
cDNArangingfrom100to5000basepairscanbeimmobilizedtothe
microarraysurfaceusingdirectroboticspotting.
Spotting is usually achieved by pin contact or drop dispense: Spottingisusuallyachievedbypincontactordropdispense:
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DNAchip Affymetrix
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Photolithography
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Photolithography
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Oligonucleotidearray
immobilized
oligonucleotide
probes showing probesshowing
hybridizationof
unknowntoa
specificprobe.
probesare
arrangedinplanar g p
arrays.
the hybridized thehybridized
regionscanbe
detectedby
fluorescence.
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DNAarray
EveryonesDNAis99.9%
identical
SmalldifferencesinDNA
sequence can have large effect sequencecanhavelargeeffect
onhealthanddisease.
Smallmutationinagenecan
k causeittonotwork.
Researchersusemicroarrays
to determine the sequence for todeterminethesequencefor
thousandsofsinglenucleotide
polymorphisms(SNPs)inthe
genome.
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www.affymetrix.com
20
DNAarray
FourbasesinDNA:adenine(A),
guanine(G),thymine(T),
cytosine(C)
C pairs with G A pairs with T CpairswithG,ApairswithT
Twocomplementarystrandscan
fittogether.
Evenasinglebasethatdoesnt
matchitspartnercouldkeepone
single strand from binding to singlestrandfrombindingto
anothersinglestrand.
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DNAarray
DNAmicroarrayusethis
hybridizationtoidentifyeach
SNPgenotype.
Inthisexample,theSNPcan
betheC/Ggenotypeorthe
/ T/Agenotype.
Arraycanidentify100,000
SNPs or more SNPsormore.
The10karrayhas>400,000
features.
Eachfeature>8mdiameter.
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DNAarray
Extract DNA. ExtractDNA.
MakecopiesoftheDNA.
FragmentDNAchains
intoshortpieces. p
Attachbiotintoeach
strand strand.
Biotinisusedlaterto
attachfluorescent
markers markers.
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DNAarray
WashDNAsampleover
array.
Ifthesequenceofbases
in the sample matches inthesamplematches
thatofaDNAprobe,the
twostrandswill
hybridize.
RinsearraysothatDNA
thatdidntpairiswashed
away away.
ThehybridizedDNAis
tagged with biotin taggedwithbiotin.
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DNAarray
Fluorescent stain Fluorescentstain
washedoverthe
array.
In this example the Inthisexample,the
DNAinthesample
matchedtheATTCATG
probe.Thispersonhas p p
theC/Ggenotypefor
thatSNP.
Canmeasuremultiple
SNPgenotypesat
once once.
EECE432 2011 KCheung 25
Geneexpressionchip
Humanexpressionarrays p y
have>1.3millionprobes
todetect50,000different
RNAsequences.
Fluorescencecoming
fromtheprobelocations
indicateswhetheragene
isexpressedornot.
Canmeasurehowmuch
ofanRNAwaspresentin
thesample.
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Opticalbiosensors
Transductionprinciples:
l b b colorabsorbance
fluorescence
plasmons(nanoparticlesandsurface)
chemiluminescence
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Fluorochromes
Whenafluorochromeisexcited
l h h atanappropriatewavelength,the
electronsjumpfromtheground
statetoahighervibrationalstate
(femtoseconds). (femtoseconds).
Theseelectronsdecaytothe
lowestexcitedstate
(picoseconds).
Theythendecaymoreslowly
(nanoseconds)backtothe
groundstateandemitaphoton
whosewavelengthislongerthan
the exciting wavelength
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theexcitingwavelength.
28
Fluorescencefilter
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Fluorescentdyes
Themaximumexcitationandemission
wavelengthsofseveralcommonlyused
fluorescentdyesareshowninrelationtothe y
correspondingcolorsofthespectrum.
Thephotonemittedbyadyemoleculeis
necessarilyoflowerenergy(longerwavelength)
h h h b b d d h f thanthephotonabsorbedandthisaccountsfor
thedifferencebetweentheexcitationand
emissionpeaks.
GFP is a green fluorescent protein not a dye GFPisagreenfluorescentprotein,notadye.
DAPIiswidelyusedasageneralfluorescentDNA
dye,thatabsorbsUVlightandfluorescesbright
blue.
Theotherdyesareallcommonlyusedto
fluorescentlylabelantibodiesandotherproteins.
EECE432 2011 KCheung 30
Summary DNAarrays
applications
fabricationmethods
fluorescenceasanopticaldetectionmethod
Multiplefluorescentprobe
microscopy
Cellinmitosis:
spindlemicrotubules green
fluorescent antibody fluorescentantibody
centromeres redfluorescent
b d antibody
DNA bluefluorescentdye
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2.DNAseparation
1. conventionalseparationbygelelectrophoresis p y g p
electrophoresis
2. separationinmicroscaledevices
electrokineticeffects:electrophoresis+electroosmosis p
LearningObjectives
Attheendofthissectionthestudentshouldbeableto:
explainhowDNAmoleculesmovethroughagelmatrix,thatthemolecules
areseparatedbysize,andwhyDNAmovestowardthepositivepole
define electrophoresis and describe how the electrophoretic velocity is defineelectrophoresisanddescribehowtheelectrophoreticvelocityis

electrophoretic
andE.
defineelectroosmosisanddescribehowtheelectroosmoticvelocityis

electro osmotic
and E.
electroosmotic
andE.
describecapillaryelectrophoresis,thescalingofseparationefficiency,and
itsresolutionlimits
compare the different separation methods: slab gel electrophoresis
EECE432 2011 KCheung
comparethedifferentseparationmethods:slabgelelectrophoresis,
capillaryelectrophoresis
32
DNAinsolution
p
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DNAseparationbygelelectrophoresis
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Electrophoresis
Electrophoresis themovementofanelectrically
chargedsubstanceundertheinfluenceofanelectric
fi ld field.
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Gelelectrophoresis
Thetypesofgelmostcommonlyusedfor
DNA l h i DNAelectrophoresis:
Polyacrylamidegels
separationof10 500bp(0.17m)
Agarosegels
separation of 300 100 000 bp (34 m) separationof300100,000bp(34m)
Gelshaveconventionallybeenrunina
"slab"format.
Aftertheseparationiscompleted,the
fractionsofDNAfragmentsofdifferent
lengthareoftenvisualizedusinga
fluorescent dye specific for DNA such as fluorescentdye specificforDNA,suchas
ethidiumbromide.Thegelshowsbands
correspondingtodifferentDNAmolecules
populationswithdifferentmolecularweight.
Fragmentsizedeterminationistypically
donebycomparisontocommercially
availableDNAladders containinglinearDNA
fragmentsofknownlength(10base
)
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pairs=3.4microns)
36
PolyacrylamideGelElectrophoresis(PAGE)
Electrophoresisisperformedinathin,verticalslabofpolyacrylamide.The
directionofflowisfromtoptobottom.
Thesievingactionofaporouspolyacrylamidegelseparatesmolecules
accordingtosize,withthesmallestmovingmostrapidly.
Molecules that are small compared with the pores in the gel readily move Moleculesthataresmallcomparedwiththeporesinthegelreadilymove
throughthegel,whereasmoleculesmuchlargerthantheporesarealmost
immobile.Intermediatesizemoleculesmovethroughthegelwithvarious
degreesoffacility.
EECE432 2011 KCheung
Biochemistry, Berg, Tymoczko, and Stryer
37
Gelelectrophoresis
SeparateDNAmoleculesbysize.
Intheseexamples,electrophoresisisfromtopto
bottom,sothatthelargestandthusslowest
movingDNAmoleculesarenearthetopofthegel.
In(A)apolyacrylamidegelwithsmallporesisused ( ) p y y g p
tofractionatesinglestrandedDNA.Inthesizerange
10to500nucleotides,DNAmoleculesthatdifferin
sizebyonlyasinglenucleotidecanbeseparated
fromeachother.SincetheDNAmoleculesusedin
these reactions are radiolabeled their positions can thesereactionsareradiolabeled,theirpositionscan
bedeterminedbyautoradiography,asshown.
In(B)anagarosegelwithmediumsizedporesis
usedtoseparatedoublestrandedDNAmolecules.
This method is most useful in the size range 300 to Thismethodismostusefulinthesizerange300to
10,000nucleotidepairs.Theyhavebeendetected
bytheirfluorescencewhenstainedwiththedye
ethidiumbromide.
In(C)thetechniqueofpulsedfieldagarosegel ( ) q p g g
electrophoresis hasbeenusedtoseparate16
differentyeastchromosomes,whichrangeinsize
from220,000to2.5millionnucleotidepairs.DNA
moleculesaslargeas10
7
nucleotidepairscanbe
t d i thi
EECE432 2011 KCheung
separatedinthisway.
Molecular Biology of the Cell, Alberts et al.
38
Electrokineticeffects
studyofthemotionsofionizedparticlesor
moleculesandtheirinteractionswithelectricfields
andthesurroundingfluid
electroosmosis fluidmovesrelativetoastationary
chargedorconductingsurfacethroughapplicationofan
electricfield.
electrophoresis charged species in a fluid are moved by electrophoresis chargedspeciesinafluidaremovedby
anelectricfieldrelativetothefluidmolecules.
dielectrophoresis dielectricparticlesexperienceanet
forceinaspatiallynonuniform electricfield.
EECE432 2011 KCheung 39
Electrokinetics:
Section4.3.3,BiomedicalMicrosystems,EllisMeng
Electricaldoublelayer
Atthesolidliquidinterfaceinanaqueouselectrolytesolution,thereisan
accumulationofthechargecarriers.Thechargecarrierslocatedatthe
surfacearecompensatedbycounterions thatarepartlyfixed(Sternlayer)
andpartlyinadiffusedistribution. p y
shearplane boundarybetweencompactlayeranddiffuselayer.
Example:inglassthereareunbalancedSiO

terminations attractionof
positiveions.
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ZetaPotential
i th t t ti l
The zeta potential is the potential value at the outside of the
is the zeta potential
Thezetapotentialisthepotentialvalueattheoutsideofthe
immobilizedions(shearplane).
Onlynonimmobilizedionsparticipateinelectrokinetic
effects effects.
TheDebyelength
D
isacharacteristiclengthofelectrostatic
screening(diffuselayer).Inthislayer,iondensityisthus
unbalanced unbalanced.
Debyelengthdependsnotdirectlyonthesurfacecharges,but
ontheelectrolytecharacteristics(ionchargeanddensity).
Surface charges (i e zeta potential) depend on the pH of the Surfacecharges(i.e.zetapotential)dependonthepHofthe
solution.
EECE432 2011 KCheung 41
Debyelength
k T
0
2
2
r B
D
A
k T
N e I

=
2
1
2
i i
I c z =

EECE432 2011 KCheung 42
Electroosmosis(Electroosmoticflow,EOF)
a flow is generated by the mobile ions (diffuse layer) aflowisgeneratedbythemobileions(diffuselayer)
attheinterfacebyanappliedelectricfield.
EECE432 2011 KCheung 43
EOF
On a plane surface Onaplanesurface,
decreasesexponentiallywithin
thedoublelayerandvanishes
f f it farfromit.
Outsideofthedoublelayer,
wherepotentialiszero:

E
u
r 0
=
(HelmholtzSmoluchowski equation)

0
EECE432 2011 KCheung 44
Biomoleculeseparationtechniques
Chromatography
Liquid(andgas)chromatographyisaseparationmethodthatexploitsthedifferencesinpartitioning
behaviorbetweenamobilephaseandastationaryphasetoseparatethecomponentsinamixture.
Componentsofamixturemaybeinteractingwiththestationaryphasebasedoncharge,relative
solubilityoradsorption.
HPLC
Highperformanceliquidchromatography(orhighpressureliquidchromatography,HPLC)isaformof
columnchromatographyusedfrequentlyinbiochemistryandanalyticalchemistry.Theanalyteis
forcedthroughacolumnofthestationaryphaseinaliquid(mobilephase)athighpressure,which
d h h d h h d h h decreasesthetimetheseparatedcomponentsremainonthestationaryphaseandthusthetime
theyhavetodiffusewithinthecolumn.
Thisleadstonarrowerpeaksintheresultingchromatogramandbetterresolution(it'seasierto
differentiateonepeakfromanother)andsensitivity(tall,narrowpeakscanbeeasiertodiscriminate
fromnoisethanshorter,broaderpeaks). , p )
Capillaryelectrophoresis
Capillaryelectrophoresis(CE)canbeusedtoseparateionicspeciesbytheirchargeandfrictional
forces.Intraditionalelectrophoresis,electricallychargedanalytesmoveinaconductiveliquid
di d h i fl f l i fi ld mediumundertheinfluenceofanelectricfield.
Duetoitshighefficiencyandcapacityforrelativelyhighresolution,capillaryelectrophoresiscanbe
usedtoseparateanddetermineawidevarietyofcompoundsincludingsimpleinorganicions,metal
ions,oligosaccharides,nucleicacids,andproteins.CEismostcommonlyused,however,toanalyze
larger,watersolublebiomolecules.
EECE432 2011 KCheung
g ,
45
CapillaryElectrophoresis
Separationofmoleculesbycharge
EECE432 2011 KCheung 47
Onchipseparation
1. Injection
2 Separation 2. Separation
EECE432 2011 KCheung 48
Glasschips microfabrication
EECE432 2011 KCheung
Z.H.FanandD.J.Harrison,"Micromachiningofcapillaryelectrophoresisinjectorsandseparatorsonglasschips
andevaluationofflowatcapillaryintersections,"AnalyticalChemistry,vol.66,pp.177184,1994.
49
Sampleinjectionandseparation


(1) (2)
Sample introduction




(3) (4)
S l i j t d S ti
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Sample injected Separation
50
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R. Bharadwaj, J. G. Santiago, and B. Mohammadi, "Design and optimization of on-chip capillary
electrophoresis," Electrophoresis, vol. 23, pp. 2729-2744, 2002.
51
Resolutionlimitations
Resolutionlimitationduetoinjectionpluganddetectionsize
Totalpeakvariance:
EECE432 2011 KCheung 53
DNAseparation
E = 204 V/cm
l = 13 mm l = 13 mm
N ~ 1.7 x 10
5
acrylamide-based sieving matrix
channels: 35 m wide, 12 m deep
separation of a 100-bp DNA ladder
EECE432 2011 KCheung
BOUSSE, L., et al (2000) Electrokinetically Controlled Microfluidic Analysis Systems. Annual
Review of Biophysics and Biomolecular Structure, 29, 155-181.
54
Summary DNAseparation
Electrophoresis the movement of an electrically charged substance under the Electrophoresis themovementofanelectricallychargedsubstanceunderthe
influenceofanelectricfield.
apowerfulmeansofseparatingproteinsandothermacromolecules,suchasDNAand
RNA.
ConventionalDNAseparationbygelelectrophoresis themovementofan
electricallychargedsubstanceundertheinfluenceofanelectricfield
Thesievingactionof
aporousgel
separatesmolecules
accordingtosize,
withthesmallest
movingmostrapidly.
EECE432 2011 KCheung 55
3.DNAsequencingandamplification
identification of the identificationofthe
nucleotidesequencein
aDNAchain.
theremustbesufficient
DNApresent,andthisis
obtainedfromeither:
abacterialcloned
fragment OR fragment,OR
aPCRamplified
sequence.
EECE432 2011 KCheung 56
3.DNAsequencingandamplification
Learning Objectives LearningObjectives
Attheendofthissectionthestudentshouldbeableto:
d fi th t DNA i definethetermDNAsequencing
describethestepsofthesequencingmethod
definePCR
namethestepsthattakeplaceduringaPCRcycleand
th diti i hi h th t theconditionsinwhichthestepsoccur
compareconventionalPCRwithmicroscalePCRsystems
comparetimedomainPCRandspacedomainPCR
EECE432 2011 KCheung 57
DNAsequencing
Introduceasmallconcentrationofdideoxynucleotides(ddNTPs)ofonetypeto
thereactionmixture(ddA,ddC,ddG,orddT).Thesespecialnucleotidesterminate
polymeraseduplicationwhencaptured.
Thereactionthusgeneratescomplementarystrandfragmentsterminatingatall
possiblepositionsofthematchingdideoxide.
Synthesisisrepeated4timeswiththe4differentterminationsites(whichare
EECE432 2011 KCheung
y p (
labelledindifferentcolors)
The4replicatedsamplesareruninparallelinagel.
58
EECE432 2011 KCheung 59
DNAsequencing
Replicationisstoppedby
nucleotideanalog(ddNTP)
specific to each base type specifictoeachbasetype
(AanalogwillstopatAsites,
TanalogwillstopatTsite,
etc ) etc)
Synthesisisrepeated4
timeswiththe4different
termination sites (which are terminationsites(whichare
labelledindifferent
colors)
h l d l The4replicatedsamples
areruninparallelinagel.
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AutomatedDNAsequencing
EECE432 2011 KCheung 61
DNAsequencing
ThegenomesequencedbytheInternational
HumanGenomeSequencingConsortium
(actuallyacompositefromseveral ( y p
individuals)took13yearsandcost$3billion
(2000).Now,usingthelatestsequencers
fromIllumina,ofSanDiego,California,a
h b d i i ht d t humangenomecanbereadineightdaysat
acostofabout$10,000.Noristhattheend
ofthestory.AnotherCalifornianfirm,Pacific
Biosciences,ofMenloPark,hasatechnology
thatcanreadgenomesfromsingleDNA
molecules.Itthinksthatinthreeyearstime
thiswillbeabletomapahumangenomein
15 minutes for less than $1 000 And a rival
Aspecialreportonthehumangenome:
Biology2.0|TheEconomistJun17th2010
15minutesforlessthan$1,000.Andarival
technologybeingdevelopedinBritainby
OxfordNanopore Technologiesaspiresto
similarspeedsandcost.
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PCR
EECE432 2011 KCheung 63
ComponentsrequiredforPCR
Primers
DNApolymerase
Anadequatesupplyofnucleotides
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PCR thermalcycling
EECE432 2011 KCheung 65
PCR
EECE432 2011 KCheung
Molecular Biology of the Cell, Alberts et al.
66
HowPCRisusedinforensicscience
ThestartingmaterialforaPCRcanbeasinglehairoratinysampleofbloodthat
l f h i wasleftatthecrimescene.
TheDNAcontainsrunsofshort,repeatedsequences,suchasCACACA,whichare
foundinvariouspositions(loci)inthehumangenome.Arunofrepeated
nucleotidesofthistypeiscommonlyreferredtoasahypervariablemicrosatellite
sequence also known as a VNTR (variable number of tandem repeat) sequence sequencealsoknownasaVNTR(variablenumberoftandemrepeat) sequence.
Intheschematicexampleshownhere,thesamethreeVNTRlociareanalyzed
(requiringthreedifferentpairsofspeciallyselectedoligonucleotideprimers)from
threesuspects(individualsA,B,andC),producingsixDNAbandsforeachperson
after polyacrylamide gel electrophoresis afterpolyacrylamidegelelectrophoresis.
Althoughsomeindividualshaveseveralbandsincommon,theoverallpatternis
quitedistinctiveforeach.Thebandpatterncanthereforeserveasafingerprint
toidentifyanindividualnearlyuniquely.
Thefourthlane(F)containstheproductsofthesamereactionscarriedoutona
forensicsample.Whenexaminingthevariabilityat5to10differentVNTRloci,the
oddsthattworandomindividualswouldsharethesamegeneticpatternbychance
canbeapproximatelyonein10billion.
EECE432 2011 KCheung 67
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EECE432 2011 KCheung 68
PCR
ThePCRprocessusuallyconsistsofaseriesoftwenty
tothirtyfivecycles.
microlitervolumesofDNAarehandledinvialsand
manuallyloadedintodesktopthermalcyclersandgel
separators separators.
typicalsequencingprotocolrequires68hours
costofseveralhundreddollars
scalingdown:
increaseinthroughput
reducedanalysistimes
reducedreagentcosts
EECE432 2011 KCheung 69
MicroscalePCRchips
PerformingPCRonamicrochipcanbeclassifiedintoeithera
timedomainoraspacedomainapproach:
TimedomainPCR Inthisformatthereactionmixtureiskept
stationaryandthetemperatureofthesurroundingreaction
chamberiscycledbetweenthedifferenttemperatures.
SpacedomainPCR Inthisformatthereactionmixtureis
movedbetweendifferentfixedtemperaturezones.
EECE432 2011 KCheung 70
TimedomainPCR
thereactionmixtureiskeptstationaryandthetemperatureof
h d h b l d b h thesurroundingreactionchamberiscycledbetweenthe
differenttemperatures.
Due to its small thermal mass, the structure can be heated at Duetoitssmallthermalmass,thestructurecanbeheatedat
ratesof15/sandcycletimesof~1min.Atwentycycle
amplificationina50microliter wellwascarriedout~4times
fasterthaninaconventionalcyclerwithamuchlowerpower
EECE432 2011 KCheung
Mastrangelo et al., Proceedings of the IEEE, August 1998, 1-15.
budget.
71
MicrofabricatedPCRreactors
EECE432 2011 KCheung 72
SpacedomainPCR continuousflow
In this format the reaction Inthisformatthereaction
mixtureismovedbetween
differentfixedtemperature
zones. zones.
Thefluidsarepumpedby
h d t ti th hydrostaticpressure;the
channeldimensions(40m
by90m)weresuchthata
d f 1 b pressuredropof1barover
thewholechipresultsina
flowthroughtimeof4min
f h 20 l forthe20cycles.
EECE432 2011 KCheung
Chemical Amplification: Continuous-Flow PCR on a Chip, Kopp et al., SCIENCE (1998),vol 280
73
SpacedomainPCR fluidiccontrol
Multilayersoftlithographyisusedtomake y g p y
3Dmonolithicelastomerdeviceswitha
combinationofairandfluidchannels.
Whenanairchannelpassesaboveanother
fluidchannel,thethinmembranebetween
thesetwochannelsbecomesavalve.
Byapplyingairpressureintheairchannel,
themembranecollapsesandstopsthefluid
flow.
On/offstatesofeachmicrovalve are
controlledbyexternalpneumaticvalves
whicheitherapply100kPa airpressureto
themicrovalvesorventthemtothe
atmosphere atmosphere.
Releasingthepressurethenreopensthis
valve.
Threevalvesinseriesbecomeaperistaltic
pump when an appropriate on/off air pumpwhenanappropriateon/offair
pressuresareappliedinasequence.
Theloopisthecomponent,atwhichthe
DNAhybridizationprobesarelaiddown
along the ring on top of the glass substrate
EECE432 2011 KCheung
alongtheringontopoftheglasssubstrate.
74
SpacedomainPCR rotaryflow
DNAsamplesandlater
fluorescent intercalating dyes can
denaturation
fluorescentintercalatingdyescan
enterfromtwobranchesofaT
channel.
Threeheatersarearrangedinthe
f ll d ( l k f
annealing
extension
followingorder(clockwisefrom
upperright):denaturation,
annealing,andextension.
Assemblyoftherotarymicrochip y y p
andtheheaters:
RTVblockwithcontrolchannels
atthebottomsurface
thin RTV layer with fluid channel thinRTVlayerwithfluidchannel
atthebottomsurface
glasscoverslip;heaterswith
leads(sputteredontoaglass
slide).
EECE432 2011 KCheung
)
Electrophoresis 2002, 23, 15311536
75
Summary DNAamplification
forDNAsequencing,theremustbesufficientDNA
present p
PCR a molecular biological technique for amplifying PCR amolecularbiologicaltechniqueforamplifying
(creatingmultiplecopiesof)DNA.
alsousedinforensicscience
microscalePCR
EECE432 2011 KCheung 76