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METHODARTICLE

Parallel DNA polymerase chain reaction: Synthesis of two


different PCR products from a DNA template [v1; ref
status: indexed, http://f1000r.es/4sm]
VikashBhardwaj1,KulbhushanSharma2
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version1

published
31dic2014

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Conventionally,inapolymerasechainreaction(PCR),oligonucleotideprimersbindtothetemplate
DNAinanantiparallelcomplementarywayandthetemplateDNAisamplifiedasitis.Herewe
describeanapproachinwhichthefirstprimerbindsinaparallelcomplementaryorientationtothe
singlestrandedDNA,leadingtosynthesisinaparalleldirection.Furtherreactionshappenedina
conventionalwayleadingtothesynthesisofPCRproducthavingpolarityoppositetothetemplate
used.ThisisthefirststudyshowingthatsynthesisofDNAcanhappenalsoinaparalleldirection.
WereportthatfromasinglestrandedDNAtemplate,twodifferentbutrelatedPCRproductscanbe
synthesized.

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Grantinformation:Theauthor(s)declaredthatnograntswereinvolvedinsupportingthiswork.

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1 HarishkumarMadhyastha,MiyazakiMedical
College,Japan

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2 RamGopal,UppsalaUniversity,Sweden
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Correspondingauthor:VikashBhardwaj
Howtocite:Bhardwaj V and Sharma K. Parallel DNA polymerase chain reaction: Synthesis of two different PCR
products from a DNA template [v1; ref status: indexed, http://f1000r.es/4sm]F1000Research2014,3:320(doi:
10.12688/f1000research.5813.1)

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RESEARCHARTICLE

REVISED Characterization of M-laurdan, a


versatile probe to explore order in lipid
membranes [v2; ref status: indexed,
http://f1000r.es/4on]

Copyright:2014BhardwajVandSharmaK.ThisisanopenaccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicence,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedthe
originalworkisproperlycited.DataassociatedwiththearticleareavailableunderthetermsoftheCreativeCommons
Zero"Norightsreserved"datawaiver(CC01.0Publicdomaindedication).
Competinginterests:Nocompetinginterestsweredisclosed.

METHODARTICLE

Firstpublished:31dic2014,3:320(doi:10.12688/f1000research.5813.1)
Firstindexed:19ene2015,3:320(doi:10.12688/f1000research.5813.1)
Latestpublished:31dic2014,3:320(doi:10.12688/f1000research.5813.1)

Follow-up: Prospective compound design using


the SAR Matrix method and matrix-derived
conditional probabilities of activity [v1; ref status:
indexed, http://f1000r.es/56v]

Introduction
OurfundamentalknowledgeofDNAstructureisbasedontheWatsonCrickmodelofDNAdoublehelix,inwhichtwo
polynucleotidechainsrunninginoppositedirectionareheldtogetherbyhydrogenbondsbetweenthenitrogenous
bases.Guaninecanbindspecificallyonlytocytosine(GC)whereasadeninecanbindspecificallytothymine(AT).
Thesereactionsaredescribedasbasepairingandthepairedbasesaresaidtobecomplementary1.Conformational
polymorphismofDNAisnowextendingbeyondtheWatsonCrickdoublehelix.In1986,usingforcedfieldcalculationfor
ashortATrichDNA,PattabiramanproposedthehypothesisthathomopolymericduplexDNAcontainingd(A)6d.(T)6
canformathermodynamicallystableparallelrighthandedduplexDNAwithreverseWatsonCrickbasepairing.Healso
reportedthatthenumberandtypeofhydrogenbondsbetweenATbasepairarethesameasthatofantiparalleldouble
helix2.In1988,theexperimentalstrategiesbyRamsingandJovinconfirmedthatDNAcontainingATbasepairscan
existasastableparallelstrandedhelix.TheTmvalueofbothPSDNA(parallelstrandedDNA)andAPSDNA
(antiparallelstrandedDNA)showedaclassicaldependenceuponsaltconcentration.Theyreportedthatatanygiven
NaClconcentration,themeltingtemperatureofPSDNAwas15ClowerthanitsAPSDNAcounterpart.In2mMMgCl2,
themeltingtemperatureforPSDNAandAPSDNAwasreportedapproximatelysameasthoseobtainedin0.20.3M
NaCl,demonstratingpronouncedstabilizationaffordedbydivalentcations3.AsimilarstudybySandeetal.onhairpin

CORRESPONDENCE

Activity artifacts in drug discovery and different


facets of compound promiscuity [v1; ref status:
indexed, http://f1000r.es/4gz]

Browse by related Topics

Genomics
MacromolecularChemistry

deoxyoligonucleotideshavingoligonucleotidessequenceinparallelpolarities(PShairpin)alsoconfirmedtheexistence
ofparallelstrandedconformation.Theyhaveshownthatparallelstrandedhairpinsformstableduplexandget
denaturedat10ClowerthancorrespondingAPSoligomers4.Thesetwoexperimentalstudiesprovidedevidencethat
DNAcontainingATbasepairscanformbothPSDNAandAPSDNA.In1992,Tchurikovetal.showedthatparallel
complementaryprobesofnormalnucleotideconsistingofbothAT/GCbasepairscanbeusedformolecular
hybridizationexperiments,indicatingthestabilityofGCcontainingparallelDNA5.In1993,Borisovaetal.reportedthat
GCpairsina40basepairparallelduplexDNA(consistingofnaturalDNAsequence)aremorethermostablethanAT
basepairs6.Furthermore,othersimilarreportshaveshownthattherearenodrasticdifferencesinnearestneighbor
basepairinteractionsbetweenPSDNAandAPSDNAhavingmixedAT/GCcomposition7.Thespecificityofthe
interactionbetweenthestrandsinparallelDNAhasalsobeenstudiedanditissohighthatparallelprobeasshortas40
nucleotidelengthisabletodetectaspecificbandinSouthernblothybridizationsonwholegenomeDNA8.The
polymerasechainreaction(PCR)developedbyMullisconsistsofdenaturationofdoublestrandedDNA,primer

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annealingandextension.TheprocessisrepeatedmultipletimesandthetemplateDNAisamplifiedmillionsoftimes
withoutanychangeinpolarityofDNA9(Figure1).In2000,VeitiaandOttolenghireportedthatseveralattemptsto
amplifyL15253byPCRusingdifferentpairsofprimerswereunsuccessful.Theysuggestedthatthereareno
thermodynamicconstraintswhichwillpreventparallelnucleicacidsynthesis,andthedeoxynucleotidetriphosphates
usedforanormalantiparallelpolymerizationreactioncanalsoserveforaparallelreaction,providedthatthe
polymeraseenzymeiscapableincatalyzingthenucleophilicinteractionbetweenthe3OHanda5PPPfrom
nucleotidesarrangedinaparallelwaywithrespecttothetemplateDNA10.

Figure1.SchematicdiagramshowingPCRamplificationofasinglestranded
DNAbyusingconventionalantiparalleloligonucleotideprimers.
DownloadasaPowerPointslide

Inthisstudy,weexploredwhetherparallelDNAsynthesisisfeasible.Weproposedthehypothesisthatthisreactioncan
bepossibleifwestartareactionusingsinglestrandedDNAasatemplate.WehaveshownthattheTaqDNA
polymerasecanevenextendtheoligonucleotideprimerannealedtosinglestrandedDNAinaparallelcomplementary
manner.ThedetailsofhowourproposedparallelDNAPCRdiffersfromtheconventionalPCRisshowninFigure1and
Figure2.

Figure2.SchematicdiagramshowingPCRamplificationofasinglestranded
DNAbyusingthePDPCR(parallelDNAPCR)approachinwhichthefirst
primerbindstothetemplateDNAinaparallelcomplementarymanner.
ThesecondprimerbindstothenewlysynthesizedDNAinanantiparallelmanner
andlaterbothprimersamplifythenewDNAinaconventionalmanner.PCR
productsobtainedwillhaveoppositepolarityascomparedtothetemplateused.
DownloadasaPowerPointslide

Materials and methods


PCR
PAGEpurifiedsinglestrandedDNAof120bpwascommerciallyobtainedatascaleof1O.D.fromSigmaAldrich,USA.
PCRoligonucleotideprimerswerealsopurchasedatascaleof0.05O.D.fromSigmaAldrich.Thesequenceofcustom
synthesizedtemplateDNAandoligonucleotideprimersusedinthestudyareshowninTable1.InthePDPCRreaction,
weused(PDPCR1)and(PDPCR2)primersetwhileforconventionalPCRweused(PCR1)and(PCR2)primers
(seeTable1).Restofthereactionremainedsame.ThedetailsofPCRreactionmixwereasfollows:totalreaction
mix=50l,primers=1leach(50picomole),TaqDNApolymerase=0.5l(5U/l),dNTPmix=0.5l(10mM),10XPCR
buffer=5l,water=39landtemplateDNA=3l(0.114ng).TaqDNApolymerase(M0273S)anddNTPmix(N0447S)
werepurchasedfromNEB(NewEnglandBiolabs).PCRanalysiswasperformedusingVeriti ThermalCycler(Applied
Biosystem)bytakingsinglestrandedtemplateDNAandamplifyingitfor30cyclesatvaryingannealingtemperatureviz.
45C,50C,55C,58C,60C,65C.PCRprogrammingincluded30cyclesofdenaturationat95Cfor15seconds,
annealingatvaryingtemperaturesfor30seconds(asexplainedabove)andextensionat72Cfor30seconds.

Sequence

5GCGCG

Oligonucl

(PCR1)5

Oligonucl

(PDPCR1

Table1.ShowssequenceofcustomsynthesizedtemplateDNAand
oligonucleotideprimersusedinthisstudy.

Agarose gel electrophoresis


PCRproductsobtainedintworeactionswereseparatedon1%agarosegelcontainingethidiumbromideandwererun
andobservedingeldoc(DNRBioimagingsystem,Jerusalem,Israel)underUV.Electrophoresisapparatususedin
theseexperimentswaspurchasedfromChromusBiotech,Bengaluru,Indiawhereaschemicals{Agarose(A9539)and
EtBr(E7637)}werepurchasedfromSigma,USA.

Sequencing
TheamplifiedproductsweresequencedatEurofinsGenomicsIndiaPvt.Ltd.KarnatakaIndia.

Real time PCR


RealtimePCRwasperformedwith2XSYBRGreenmastermix(K0221,ThermoScientific,Pittsburgh,USA).Thedetails
ofrealtimePCRreactionmixwereasfollows:totalreactionmix=10l,primers=0.25leach,(50picomole),2XSYBR

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ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

greenmastermix=5l,water=4landtemplate=0.5l(1:1000dilutionfromoriginalstockof0.96picomole).InthePD
PCRreaction,weused(PDPCR1)and(PDPCR2)primersetwhileforconventionalPCRweused(PCR1)and
(PCR2)primers(seeTable1).NegativecontrolincludedreactionmixwithouttemplateDNA.Reactionswereincubated
at94Cfor5minutes,followedby30PCRcyclesof94Cfor15seconds,50Cfor30secondsand72Cfor60seconds
usingMx3005PqPCRSystemAgilentTechnologies,Inc.Thedatawereanalyzedbyusing2Ct method.Theproducts
werealsorunonagarosegelandvisualisedongeldocsystemaspreviouslydescribed.

Result and discussion


Dataset1. DNAsequencingfileforPCRandPDPCR.DNAsequencingfile(DNAsequencingfile
forPCR.ab1)confirmingsinglestrandedtemplateDNAwasamplifiedasitiswhileDNAsequencingfile(DNA
sequencingfileforPDPCR.ab1)confirmingthatsinglestrandedtemplateDNAwasamplifiedasperour
proposedPDPCRschemewithinthismanuscript.
Downloadthedata

Dataset2. RealtimePCRfileforPCRandPDPCR.RealtimePCRamplificationofsinglestranded
DNAasperconventionalPCRandPDPCR.1Datafile
Downloadthedata

ThethermaldenaturationanalysisofparallelDNAhasshownthatitmeltsatalowertemperaturethanthe
correspondingantiparallelstructure3,4.ThisfindinggivesusthecluethatusingdoublestrandedantiparallelDNAasa
templateforPDPCRwillnotbepossibleasduringannealingsteps,antiparalleldoublestrandedDNAwillannealto
itselfwithoutbindingtoparallelstrandedcomplementaryprimers.Toavoidthis,westartedourPCRwithasingle
strandedDNAtemplate.DetailsonhowourproposedparallelDNAPCR(PDPCR)differsfromconventionalPCRare
showninFigure2.Thefirstoligonucleotideprimer(PDPCR1)wasdesignedtobindthesinglestrandedtemplateDNA
inaparallelcomplementarymanner.Theparallelcomplementaryannealingofthefirstprimerallowedthesynthesisof
DNAinaparalleldirectiontothesinglestrandedDNAtemplate.Afterthefirstdenaturationstep,thesecond
oligonucleotideprimer(PDPCR2)wasdesignedtoannealtothenewlysynthesizedDNAinanantiparallel
complementaryorientation.Further,bothfirstandsecondprimersusedinthisreactionamplifiedthenewsecondDNA
strandinaconventionalwaybybindinginanantiparallelcomplementaryway.Figure3(lanes813)showsa120bp
PCRproductamplifiedbyparallelDNAPCRschemeatannealingtemperatureof45C,50C,55C,58C,60C,65C
respectively.Inallcases,denaturationwasperformedat95Cfor15seconds,annealingfor30secondswhileextension
at72Cfor30secondforatotalof30cycles.Similarly,asacontrolreaction,thesinglestranded120bpDNAwas
amplifiedbyconventionalPCRinwhichthefirstprimer(PCR1)boundtothetemplateDNAinanantiparallelorientation
andthesecondprimer(PCR2)annealedtothenewlysynthesizedDNAinanantiparallelorientation.Figure3,Lanes
16showsa120bpproductPCRamplifiedatannealingtemperatureof45C,50C,55C,58C,60C,65C
respectivelyusingconventionalantiparallelcomplementaryprimers.Asacontrolreaction,PDPCRwasalsoperformed
usingonlyoneofthetwoprimers.Asexpected,noPCRproductswereobtained(Figure4Alane2and3).Asacontrol
reaction,conventionalPCRandPDPCRwereperformedwithoutaddinganytemplateDNA.Asexpected,noPCR
productwasobtainedconfirmingthatnoprimerdimerwasformedduringbothconventionalPCRandPDPCR(Figure
4B).TheDNAsequencingresultsconfirmedthatDNAtemplateswereamplifiedintwodifferentPCRproducts.
ConventionalPCRamplifiedthetemplateDNAinitsoriginalorientation(Figure5A)whereasPDPCRproductsreadin
aparalleldirectiontothetemplateDNA(Figure5B).PrimersandtemplateusedtoshowfeasibilityofPDPCRtillnow
(Figure3andTable1)werefurtherusedtoperformrealtimePCR.Forthis,amastermixcontainingSYBRGreenand
othercomponents(excepttemplateandprimers)wasused.Primersandtemplatewereaddedtothemastermixtomake
afinalvolumeof10l.ACtvalueof9.26wasobtainedforconventionalPCR,23.29forPDPCRwhereas33.15was
observedinnegativecontrol(withoutaddingtemplateDNA)indicatingamplificationinbothconventionalPCRandPD
PCRreactions(Figure6).TheamplificationindicatedbyCtvalueinrealtimePCRwasalsoconfirmedbyrunningthe
productonagarosegel(Figure6,lowerpanel).WeakamplificationsinPDPCRmaybeattributedtothefactthatthe
actualamplificationinconventionalPCRisonestepaheadthanthePDPCR.Thefirstamplificationinconventional
PCRstartsasearlyasdenaturationfollowedbyannealing(Figure1).Ontheotherhand,inPDPCRanewtemplateis
firstsynthesizedduringthefirstamplification(representedbygreencolorinFigure2).Oncethetemplateisready,the
conventionalPCRgoeson.Therefore,theamplifiedproductshowslowintensityascomparedtotheconventionalPCR
products.Takingtogether,ourstudyhasshownthatDNAsynthesiscanhappeninaparalleldirectionandtwodifferent,
butrelatedPCRproductscanbesynthesizedfromthesinglestrandedtemplateDNA.Wehopethatmoremolecular
biologytechniqueswilldevelopinfuturebasedonparallelcomplementarybindingsofduplexDNA.

Figure3.PDPCR(parallelDNAPCR)andPCR:Lanes16show120bpPCR
productsamplifiedatannealingtemperatureof45C,50C,55C,58C,60C,
65C,respectively,usingconventionalantiparallelcomplementaryprimers.
Lane7is100bpmolecularweightmarkerandLanes813showPCRproductsamplifiedbyparallelDNAPCR
schemeatannealingtemperatureof45C,50C,55C,58C,60C,65C,respectively.Inallcases,denaturation
wasperformedat95Cfor15seconds,annealingfor30secondswhileextensionat72Cfor30secondfora
totalof30cycles.
DownloadasaPowerPointslide

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ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

Figure4.
ArticleNavigation
OpenPeerReview/Discussion
(A):AcontrolreactionshowingthatPCRproductswereobtainedwhenboth
primerswereaddedasperschemeinFigure2.InFigure4(A),Lane1shows120
bpPCRproductssynthesizedbyPDPCR,whileinLanes2and3,onlysingle
primerswereaddedandasexpectednoPCRproductwassynthesized.Figure
4(B)showsanegativecontrolreactionofconventionalPCRandPDPCRinwhich
thetemplateDNAwasnotadded.

PeerReviewStatus

DownloadasaPowerPointslide

Figure5.
DNAsequencingresults.Sequencingresultsin(A)showthat120bpDNAwas
amplifiedasitiswhilesequencingresultsin(B)confirmthatPCRproductswere
obtainedaspertheschemeshowninFigure2.
DownloadasaPowerPointslide

Figure6.
RealtimePCRandPDPCR(A)showampliflicationplotanddissociationcurves
obtainedafterrealtimePCRanalysisofamplificationof120nucleotidessingle
strandedtemplateDNAviaconventionalPCRandPDPCR.Incontrolreactionno
templateDNAwasadded.(B)PCRproductsobtainedinrealtimePCRwerealso
runonagarosegelandvisualisedongeldocsystem.
DownloadasaPowerPointslide

Data availability
F1000Research:Dataset1.DNAsequencingfileforPCRandPDPCR.10.5256/f1000research.5813.d4151511
F1000Research:Dataset2.RealtimePCRfileforPCRandPDPCR.10.5256/f1000research.5813.d4151612

Author contributions
VBandKSdesignedtheexperiment.VBandKScarriedouttheresearch.Bothpreparedthemanuscript.

Competing interests
Nocompetinginterestsweredisclosed.
TheF1000Researchwebsiteusescookies.Bycontinuingtobrowsethesite,youareagreeingtoouruseofcookies.Findoutmore

Grant information
Theauthor(s)declaredthatnograntswereinvolvedinsupportingthiswork.

Acknowledgments
WearethankfultoHarpreetSingh(TerritoryManager,SigmaAldrich,Gujarat,India)forprovidingthecustom
synthesizedtemplateDNAandoligonucleotideprimersusedinthisstudy.

References
1. WatsonJD,CrickFH:MolecularstructureofnucleicacidsastructureforDeoxyriboseNucleicAcid.Nature.
1953171(4356):737738.PubMedAbstract|PublisherFullText

2. PattabiramanN:CantheDoubleHelixBeParallel?Biopolymers.198625(9):16031606.PubMed
Abstract|PublisherFullText

3. RamsingNB,JovinTM:ParallelstrandedduplexDNA.NucleicAcidsRes.198816(14A):665976.
PubMedAbstract|PublisherFullText|FreeFullText

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ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

4. vandeSandeJH,RamsingNB,GermannMW,etal.:ParallelStrandedDNA.Science.1988241(4865):
551557.PubMedAbstract|PublisherFullText

5. TchurikovNA,ShchyolkinaAK,BorissovaOF,etal.:Southernmolecularhybridizationexperimentswith
parallelcomplementaryDNAprobes.FEBSLett.1992297(3):233236.PubMedAbstract|PublisherFull
Text

6. BorisovaOF,ShchyolkinaAK,ChernovBK,etal.:RelativestabilityofATandGCpairsinparallelDNA
duplexformedbyanaturalsequence.FEBSLett.1993322(3):3046.PubMedAbstract|PublisherFull
Text

7. ShchyolkinaAK,BorisovaOF,LivshitsMA,etal.:[ParallelstrandedDNAwithnaturalbasesequences].Mol
Biol.200337(2):223231.PubMedAbstract|PublisherFullText

8. TchurikovNA,ShchyolkinaAK,BorissovaOF,etal.:Southernmolecularhybridizationexperimentswith
parallelcomplementaryDNAprobes.FEBSLett.199210:297(3):2336.PubMedAbstract|PublisherFull
Text

9. MullisKB:Processforamplifyingnucleicacidsequences,UnitedStatesPatent4683202.1987.Reference
Source

10. VeitiaR,OttolenghiC:PlacingparallelstrandedDNAinanevolutionarycontext.JTheorBiol.2000206(2):
317322.PubMedAbstract|PublisherFullText

11. BhardwajV,SharmaK:Dataset1.DNAsequencingfileforPCRandPDPCR.F1000Research.2014.Data
Source

12. BhardwajV,SharmaK:Dataset2.RealtimePCRfileforPCRandPDPCR.F1000Research.2014.Data
Source

OpenPeerReview
CurrentRefereeStatus:

Version1
RefereeReport19ene2015
RamGopal,DepartmentofCellandMolecularBiology,UppsalaUniversity,Sweden
Approved

Views
173
Cite

ItisinterestingtoreadthearticleParallelDNApolymersechainreaction:Synthesisoftwodifferent
PCRproductsfromaDNAtemplatebyBhardwajandSharma.Theauthorsforthefirsttimehave
demonstratedthatprimerscanannealinparallel... Continuereading

RespondorComment

RefereeReport12ene2015
HarishkumarMadhyastha,DepartmentofAppliedPhysiology,FacultyofMedicine,Miyazaki
MedicalCollege,Japan

Views
176
Cite

Approved
Thetitleoftheresearchpaperiswelljustifiedandsuitabletothecontentofthesubject.Abstractofthe
paperiswellwrittenandconcluded.Materialsandmethod,dataanalysisanddesignoftheexperiment
arecommendable.Inmyopinion,... Continuereading

RespondorComment

DiscussthisArticle
Version1
AuthorResponse03mar2015

DrVikashBhardwaj,LovelyProfessionalUniversity,Punjab,India,India
DearReinerVeitia

Wehaveattachedtwoseparatecommunicationswereceivedearlierfromyou.
ThefirstonesaysIhavereadyourpreprintonparallelDNAwithlotofinterest.It... Continuereading

http://f1000research.com/articles/3320/v1

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ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

ReaderComment23feb2015

ReinerVeitia,France
Ihavenowhadtheopportunitytorepeatthisexperimentwiththesametemplateandparallelprimersas
describedintheMSandunfortunatelyfailedtoobtainanyamplification(at... Continuereading

AuthorResponse09feb2015

DrVikashBhardwaj,LovelyProfessionalUniversity,Punjab,India,India
CheckoutthefollowingreportpublishedatGenomewebsitebyMadeleineJohnson
"StudyClaimsPCRfromPrimersAnnealedinParallelOrientation"
CompetingInterests:Nocompetinginterestsweredisclosed.

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