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F1000.com Sign In Search Articles Channels For Authors For Referees About Blog Submit your Manuscript My Research Corresponding author: Vikash Bhardwaj Copyright: © 2014 Bhardwaj V and Sharma K. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). Views 3729 Downloads 728 Get PDF Get XML Cite Track Email Share ▬ F1000Research » Articles METHOD ARTICLE Parallel DNA polymerase chain reaction: Synthesis of two different PCR products from a DNA template [v1; ref status: indexed, http://f1000r.es/4sm] Vikash Bhardwaj , Kulbhushan Sharma Author affiliations Grant information: The author(s) declared that no grants were involved in supporting this work. Abstract Conventionally, in a polymerase chain reaction (PCR), oligonucleotide primers bind to the template DNA in an antiparallel complementary way and the template DNA is amplified as it is. Here we describe an approach in which the first primer binds in a parallel complementary orientation to the singlestranded DNA, leading to synthesis in a parallel direction. Further reactions happened in a conventional way leading to the synthesis of PCR product having polarity opposite to the template used. This is the first study showing that synthesis of DNA can happen also in a parallel direction. We report that from a singlestranded DNA template, two different but related PCR products can be synthesized. How to cite: 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] F1000Research 2014, 3:320 (doi: 10.12688/f1000research.5813.1) Competing interests: No competing interests were disclosed. First published: 31 dic 2014, 3:320 (doi: 10.12688/f1000research.5813.1) First indexed: 19 ene 2015, 3:320 (doi: 10.12688/f1000research.5813.1) Latest published: 31 dic 2014, 3:320 (doi: 10.12688/f1000research.5813.1) Introduction Our fundamental knowledge of DNA structure is based on the WatsonCrick model of DNA double helix, in which two polynucleotide chains running in opposite direction are held together by hydrogen bonds between the nitrogenous bases. Guanine can bind specifically only to cytosine (GC) whereas adenine can bind specifically to thymine (AT). These reactions are described as base pairing and the paired bases are said to be “complementary” . Conformational polymorphism of DNA is now extending beyond the WatsonCrick double helix. In 1986, using forced field calculation for a short ‘AT’ rich DNA, Pattabiraman proposed the hypothesis that homopolymeric duplex DNA containing d(A)6d.(T)6 can form a thermodynamically stable parallel righthanded duplex DNA with reverse WatsonCrick base pairing. He also reported that the number and type of hydrogen bonds between AT base pair are the same as that of antiparallel double helix . In 1988, the experimental strategies by Ramsing and Jovin confirmed that DNA containing AT base pairs can exist as a stable parallelstranded helix. The “Tm” value of both PSDNA (parallelstranded DNA) and APSDNA (antiparallelstranded DNA) showed a classical dependence upon salt concentration. They reported that at any given NaCl concentration, the melting temperature of PSDNA was 15°C lower than its APSDNA counterpart. In 2 mM MgCl , the melting temperature for PSDNA and APSDNA was reported approximately same as those obtained in 0.2–0.3 M NaCl, demonstrating pronounced stabilization afforded by divalent cations . A similar study by Sande et al. on hairpin deoxyoligonucleotides having oligonucleotides sequence in parallel polarities (PShairpin) also confirmed the existence of parallelstranded conformation. They have shown that parallelstranded hairpins form stable duplex and get denatured at 10°C lower than corresponding APS oligomers . These two experimental studies provided evidence that DNA containing “AT” base pairs can form both PSDNA and APSDNA. In 1992, Tchurikov et al. showed that parallel complementary probes of normal nucleotide consisting of both AT/GC base pairs can be used for molecular hybridization experiments, indicating the stability of GC containing parallel DNA . In 1993, Borisova et al. reported that GC pairs in a 40 base pair parallel duplex DNA (consisting of natural DNA sequence) are more thermostable than AT base pairs . Furthermore, other similar reports have shown that there are no drastic differences in nearest neighbor base pair interactions between PSDNA and APSDNA having mixed AT/GC composition . The specificity of the interaction between the strands in parallel DNA has also been studied and it is so high that parallel probe as short as 40 nucleotide length is able to detect a specific band in Southern blot hybridizations on whole genome DNA . The polymerase chain reaction (PCR) developed by Mullis consists of denaturation of doublestranded DNA, primer Open Peer Review Read the reports (2) Discuss this article Comments (3) Add a Comment Articles that may interest you RESEARCH ARTICLE Characterization of M-laurdan, a versatile probe to explore order in lipid membranes [v2; ref status: indexed, http://f1000r.es/4on] METHOD ARTICLE 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] CORRESPONDENCE Activity artifacts in drug discovery and different facets of compound promiscuity [v1; ref status: indexed, http://f1000r.es/4gz] Ο Browse by related Topics Genomics Macromolecular Chemistry Ο 1 2 1 2 2 3 4 5 6 7 8 Referee Status: Invited Referees version 1 published 31 dic 2014 1 2 report report Harishkumar Madhyastha, Miyazaki Medical College, Japan 1 Ram Gopal, Uppsala University, Sweden 2 REVISED
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  • 2/4/2015 ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

    http://f1000research.com/articles/3320/v1 1/6

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    Correspondingauthor:VikashBhardwaj

    Copyright:2014BhardwajVandSharmaK.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicence,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.DataassociatedwiththearticleareavailableunderthetermsoftheCreativeCommonsZero"Norightsreserved"datawaiver(CC01.0Publicdomaindedication).

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    METHODARTICLE

    Parallel DNA polymerase chain reaction: Synthesis of twodifferent PCR products from a DNA template [v1; refstatus: indexed, http://f1000r.es/4sm]VikashBhardwaj ,KulbhushanSharma

    Authoraffiliations

    Grantinformation:Theauthor(s)declaredthatnograntswereinvolvedinsupportingthiswork.

    AbstractConventionally,inapolymerasechainreaction(PCR),oligonucleotideprimersbindtothetemplateDNAinanantiparallelcomplementarywayandthetemplateDNAisamplifiedasitis.HerewedescribeanapproachinwhichthefirstprimerbindsinaparallelcomplementaryorientationtothesinglestrandedDNA,leadingtosynthesisinaparalleldirection.FurtherreactionshappenedinaconventionalwayleadingtothesynthesisofPCRproducthavingpolarityoppositetothetemplateused.ThisisthefirststudyshowingthatsynthesisofDNAcanhappenalsoinaparalleldirection.WereportthatfromasinglestrandedDNAtemplate,twodifferentbutrelatedPCRproductscanbesynthesized.

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

    Competinginterests:Nocompetinginterestsweredisclosed.

    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)

    IntroductionOurfundamentalknowledgeofDNAstructureisbasedontheWatsonCrickmodelofDNAdoublehelix,inwhichtwopolynucleotidechainsrunninginoppositedirectionareheldtogetherbyhydrogenbondsbetweenthenitrogenousbases.Guaninecanbindspecificallyonlytocytosine(GC)whereasadeninecanbindspecificallytothymine(AT).Thesereactionsaredescribedasbasepairingandthepairedbasesaresaidtobecomplementary .ConformationalpolymorphismofDNAisnowextendingbeyondtheWatsonCrickdoublehelix.In1986,usingforcedfieldcalculationforashortATrichDNA,PattabiramanproposedthehypothesisthathomopolymericduplexDNAcontainingd(A)6d.(T)6canformathermodynamicallystableparallelrighthandedduplexDNAwithreverseWatsonCrickbasepairing.HealsoreportedthatthenumberandtypeofhydrogenbondsbetweenATbasepairarethesameasthatofantiparalleldoublehelix .In1988,theexperimentalstrategiesbyRamsingandJovinconfirmedthatDNAcontainingATbasepairscanexistasastableparallelstrandedhelix.TheTmvalueofbothPSDNA(parallelstrandedDNA)andAPSDNA(antiparallelstrandedDNA)showedaclassicaldependenceuponsaltconcentration.TheyreportedthatatanygivenNaClconcentration,themeltingtemperatureofPSDNAwas15ClowerthanitsAPSDNAcounterpart.In2mMMgCl ,themeltingtemperatureforPSDNAandAPSDNAwasreportedapproximatelysameasthoseobtainedin0.20.3MNaCl,demonstratingpronouncedstabilizationaffordedbydivalentcations .AsimilarstudybySandeetal.onhairpindeoxyoligonucleotideshavingoligonucleotidessequenceinparallelpolarities(PShairpin)alsoconfirmedtheexistenceofparallelstrandedconformation.Theyhaveshownthatparallelstrandedhairpinsformstableduplexandgetdenaturedat10ClowerthancorrespondingAPSoligomers .ThesetwoexperimentalstudiesprovidedevidencethatDNAcontainingATbasepairscanformbothPSDNAandAPSDNA.In1992,Tchurikovetal.showedthatparallelcomplementaryprobesofnormalnucleotideconsistingofbothAT/GCbasepairscanbeusedformolecularhybridizationexperiments,indicatingthestabilityofGCcontainingparallelDNA .In1993,Borisovaetal.reportedthatGCpairsina40basepairparallelduplexDNA(consistingofnaturalDNAsequence)aremorethermostablethanATbasepairs .Furthermore,othersimilarreportshaveshownthattherearenodrasticdifferencesinnearestneighborbasepairinteractionsbetweenPSDNAandAPSDNAhavingmixedAT/GCcomposition .ThespecificityoftheinteractionbetweenthestrandsinparallelDNAhasalsobeenstudiedanditissohighthatparallelprobeasshortas40nucleotidelengthisabletodetectaspecificbandinSouthernblothybridizationsonwholegenomeDNA .Thepolymerasechainreaction(PCR)developedbyMullisconsistsofdenaturationofdoublestrandedDNA,primer

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    RESEARCHARTICLE

    Characterization of M-laurdan, aversatile probe to explore order in lipidmembranes [v2; ref status: indexed,http://f1000r.es/4on]

    METHODARTICLE

    Follow-up: Prospective compound design usingthe SAR Matrix method and matrix-derivedconditional probabilities of activity [v1; ref status:indexed, http://f1000r.es/56v]

    CORRESPONDENCE

    Activity artifacts in drug discovery and differentfacets of compound promiscuity [v1; ref status:indexed, http://f1000r.es/4gz]

    Browse by related Topics

    Genomics MacromolecularChemistry

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    version1published31dic2014

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    report report

    HarishkumarMadhyastha,MiyazakiMedicalCollege,Japan

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    RamGopal,UppsalaUniversity,Sweden2

    REVISED

  • 2/4/2015 ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

    http://f1000research.com/articles/3320/v1 2/6

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    Sequence 5 GCGCG

    Oligonucl (PCR1)5

    Oligonucl (PDPCR1

    annealingandextension.TheprocessisrepeatedmultipletimesandthetemplateDNAisamplifiedmillionsoftimeswithoutanychangeinpolarityofDNA (Figure1).In2000,VeitiaandOttolenghireportedthatseveralattemptstoamplifyL15253byPCRusingdifferentpairsofprimerswereunsuccessful.Theysuggestedthattherearenothermodynamicconstraintswhichwillpreventparallelnucleicacidsynthesis,andthedeoxynucleotidetriphosphatesusedforanormalantiparallelpolymerizationreactioncanalsoserveforaparallelreaction,providedthatthepolymeraseenzymeiscapableincatalyzingthenucleophilicinteractionbetweenthe3OHanda5PPPfromnucleotidesarrangedinaparallelwaywithrespecttothetemplateDNA .

    Figure1.SchematicdiagramshowingPCRamplificationofasinglestrandedDNAbyusingconventionalantiparalleloligonucleotideprimers.

    Inthisstudy,weexploredwhetherparallelDNAsynthesisisfeasible.WeproposedthehypothesisthatthisreactioncanbepossibleifwestartareactionusingsinglestrandedDNAasatemplate.WehaveshownthattheTaqDNApolymerasecanevenextendtheoligonucleotideprimerannealedtosinglestrandedDNAinaparallelcomplementarymanner.ThedetailsofhowourproposedparallelDNAPCRdiffersfromtheconventionalPCRisshowninFigure1andFigure2.

    Figure2.SchematicdiagramshowingPCRamplificationofasinglestrandedDNAbyusingthePDPCR(parallelDNAPCR)approachinwhichthefirstprimerbindstothetemplateDNAinaparallelcomplementarymanner.

    ThesecondprimerbindstothenewlysynthesizedDNAinanantiparallelmannerandlaterbothprimersamplifythenewDNAinaconventionalmanner.PCRproductsobtainedwillhaveoppositepolarityascomparedtothetemplateused.

    Materials and methodsPCR

    PAGEpurifiedsinglestrandedDNAof120bpwascommerciallyobtainedatascaleof1O.D.fromSigmaAldrich,USA.PCRoligonucleotideprimerswerealsopurchasedatascaleof0.05O.D.fromSigmaAldrich.ThesequenceofcustomsynthesizedtemplateDNAandoligonucleotideprimersusedinthestudyareshowninTable1.InthePDPCRreaction,weused(PDPCR1)and(PDPCR2)primersetwhileforconventionalPCRweused(PCR1)and(PCR2)primers(seeTable1).Restofthereactionremainedsame.ThedetailsofPCRreactionmixwereasfollows:totalreactionmix=50l,primers=1leach(50picomole),TaqDNApolymerase=0.5l(5U/l),dNTPmix=0.5l(10mM),10XPCRbuffer=5l,water=39landtemplateDNA=3l(0.114ng).TaqDNApolymerase(M0273S)anddNTPmix(N0447S)werepurchasedfromNEB(NewEnglandBiolabs).PCRanalysiswasperformedusingVeriti ThermalCycler(AppliedBiosystem)bytakingsinglestrandedtemplateDNAandamplifyingitfor30cyclesatvaryingannealingtemperatureviz.45C,50C,55C,58C,60C,65C.PCRprogrammingincluded30cyclesofdenaturationat95Cfor15seconds,annealingatvaryingtemperaturesfor30seconds(asexplainedabove)andextensionat72Cfor30seconds.

    Table1.ShowssequenceofcustomsynthesizedtemplateDNAandoligonucleotideprimersusedinthisstudy.

    Agarose gel electrophoresis

    PCRproductsobtainedintworeactionswereseparatedon1%agarosegelcontainingethidiumbromideandwererunandobservedingeldoc(DNRBioimagingsystem,Jerusalem,Israel)underUV.ElectrophoresisapparatususedintheseexperimentswaspurchasedfromChromusBiotech,Bengaluru,Indiawhereaschemicals{Agarose(A9539)andEtBr(E7637)}werepurchasedfromSigma,USA.

    Sequencing

    TheamplifiedproductsweresequencedatEurofinsGenomicsIndiaPvt.Ltd.KarnatakaIndia.

    Real time PCR

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

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  • 2/4/2015 ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

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    greenmastermix=5l,water=4landtemplate=0.5l(1:1000dilutionfromoriginalstockof0.96picomole).InthePDPCRreaction,weused(PDPCR1)and(PDPCR2)primersetwhileforconventionalPCRweused(PCR1)and(PCR2)primers(seeTable1).NegativecontrolincludedreactionmixwithouttemplateDNA.Reactionswereincubatedat94Cfor5minutes,followedby30PCRcyclesof94Cfor15seconds,50Cfor30secondsand72Cfor60secondsusingMx3005PqPCRSystemAgilentTechnologies,Inc.Thedatawereanalyzedbyusing2 method.Theproductswerealsorunonagarosegelandvisualisedongeldocsystemaspreviouslydescribed.

    Result and discussion

    Dataset1. DNAsequencingfileforPCRandPDPCR.DNAsequencingfile(DNAsequencingfileforPCR.ab1)confirmingsinglestrandedtemplateDNAwasamplifiedasitiswhileDNAsequencingfile(DNAsequencingfileforPDPCR.ab1)confirmingthatsinglestrandedtemplateDNAwasamplifiedasperourproposedPDPCRschemewithinthismanuscript.

    Downloadthedata

    Dataset2. RealtimePCRfileforPCRandPDPCR.RealtimePCRamplificationofsinglestrandedDNAasperconventionalPCRandPDPCR.1Datafile

    Downloadthedata

    ThethermaldenaturationanalysisofparallelDNAhasshownthatitmeltsatalowertemperaturethanthecorrespondingantiparallelstructure .ThisfindinggivesusthecluethatusingdoublestrandedantiparallelDNAasatemplateforPDPCRwillnotbepossibleasduringannealingsteps,antiparalleldoublestrandedDNAwillannealtoitselfwithoutbindingtoparallelstrandedcomplementaryprimers.Toavoidthis,westartedourPCRwithasinglestrandedDNAtemplate.DetailsonhowourproposedparallelDNAPCR(PDPCR)differsfromconventionalPCRareshowninFigure2.Thefirstoligonucleotideprimer(PDPCR1)wasdesignedtobindthesinglestrandedtemplateDNAinaparallelcomplementarymanner.TheparallelcomplementaryannealingofthefirstprimerallowedthesynthesisofDNAinaparalleldirectiontothesinglestrandedDNAtemplate.Afterthefirstdenaturationstep,thesecondoligonucleotideprimer(PDPCR2)wasdesignedtoannealtothenewlysynthesizedDNAinanantiparallelcomplementaryorientation.Further,bothfirstandsecondprimersusedinthisreactionamplifiedthenewsecondDNAstrandinaconventionalwaybybindinginanantiparallelcomplementaryway.Figure3(lanes813)showsa120bpPCRproductamplifiedbyparallelDNAPCRschemeatannealingtemperatureof45C,50C,55C,58C,60C,65Crespectively.Inallcases,denaturationwasperformedat95Cfor15seconds,annealingfor30secondswhileextensionat72Cfor30secondforatotalof30cycles.Similarly,asacontrolreaction,thesinglestranded120bpDNAwasamplifiedbyconventionalPCRinwhichthefirstprimer(PCR1)boundtothetemplateDNAinanantiparallelorientationandthesecondprimer(PCR2)annealedtothenewlysynthesizedDNAinanantiparallelorientation.Figure3,Lanes16showsa120bpproductPCRamplifiedatannealingtemperatureof45C,50C,55C,58C,60C,65Crespectivelyusingconventionalantiparallelcomplementaryprimers.Asacontrolreaction,PDPCRwasalsoperformedusingonlyoneofthetwoprimers.Asexpected,noPCRproductswereobtained(Figure4Alane2and3).Asacontrolreaction,conventionalPCRandPDPCRwereperformedwithoutaddinganytemplateDNA.Asexpected,noPCRproductwasobtainedconfirmingthatnoprimerdimerwasformedduringbothconventionalPCRandPDPCR(Figure4B).TheDNAsequencingresultsconfirmedthatDNAtemplateswereamplifiedintwodifferentPCRproducts.ConventionalPCRamplifiedthetemplateDNAinitsoriginalorientation(Figure5A)whereasPDPCRproductsreadinaparalleldirectiontothetemplateDNA(Figure5B).PrimersandtemplateusedtoshowfeasibilityofPDPCRtillnow(Figure3andTable1)werefurtherusedtoperformrealtimePCR.Forthis,amastermixcontainingSYBRGreenandothercomponents(excepttemplateandprimers)wasused.Primersandtemplatewereaddedtothemastermixtomakeafinalvolumeof10l.ACtvalueof9.26wasobtainedforconventionalPCR,23.29forPDPCRwhereas33.15wasobservedinnegativecontrol(withoutaddingtemplateDNA)indicatingamplificationinbothconventionalPCRandPDPCRreactions(Figure6).TheamplificationindicatedbyCtvalueinrealtimePCRwasalsoconfirmedbyrunningtheproductonagarosegel(Figure6,lowerpanel).WeakamplificationsinPDPCRmaybeattributedtothefactthattheactualamplificationinconventionalPCRisonestepaheadthanthePDPCR.ThefirstamplificationinconventionalPCRstartsasearlyasdenaturationfollowedbyannealing(Figure1).Ontheotherhand,inPDPCRanewtemplateisfirstsynthesizedduringthefirstamplification(representedbygreencolorinFigure2).Oncethetemplateisready,theconventionalPCRgoeson.Therefore,theamplifiedproductshowslowintensityascomparedtotheconventionalPCRproducts.Takingtogether,ourstudyhasshownthatDNAsynthesiscanhappeninaparalleldirectionandtwodifferent,butrelatedPCRproductscanbesynthesizedfromthesinglestrandedtemplateDNA.WehopethatmoremolecularbiologytechniqueswilldevelopinfuturebasedonparallelcomplementarybindingsofduplexDNA.

    Figure3.PDPCR(parallelDNAPCR)andPCR:Lanes16show120bpPCRproductsamplifiedatannealingtemperatureof45C,50C,55C,58C,60C,65C,respectively,usingconventionalantiparallelcomplementaryprimers.

    Lane7is100bpmolecularweightmarkerandLanes813showPCRproductsamplifiedbyparallelDNAPCRschemeatannealingtemperatureof45C,50C,55C,58C,60C,65C,respectively.Inallcases,denaturationwasperformedat95Cfor15seconds,annealingfor30secondswhileextensionat72Cfor30secondforatotalof30cycles.

    Ct

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  • 2/4/2015 ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

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    Figure4.

    (A):AcontrolreactionshowingthatPCRproductswereobtainedwhenbothprimerswereaddedasperschemeinFigure2.InFigure4(A),Lane1shows120bpPCRproductssynthesizedbyPDPCR,whileinLanes2and3,onlysingleprimerswereaddedandasexpectednoPCRproductwassynthesized.Figure4(B)showsanegativecontrolreactionofconventionalPCRandPDPCRinwhichthetemplateDNAwasnotadded.

    Figure5.

    DNAsequencingresults.Sequencingresultsin(A)showthat120bpDNAwasamplifiedasitiswhilesequencingresultsin(B)confirmthatPCRproductswereobtainedaspertheschemeshowninFigure2.

    Figure6.

    RealtimePCRandPDPCR(A)showampliflicationplotanddissociationcurvesobtainedafterrealtimePCRanalysisofamplificationof120nucleotidessinglestrandedtemplateDNAviaconventionalPCRandPDPCR.IncontrolreactionnotemplateDNAwasadded.(B)PCRproductsobtainedinrealtimePCRwerealsorunonagarosegelandvisualisedongeldocsystem.

    Data availabilityF1000Research:Dataset1.DNAsequencingfileforPCRandPDPCR.10.5256/f1000research.5813.d41515

    F1000Research:Dataset2.RealtimePCRfileforPCRandPDPCR.10.5256/f1000research.5813.d41516

    Author contributionsVBandKSdesignedtheexperiment.VBandKScarriedouttheresearch.Bothpreparedthemanuscript.

    Competing interestsNocompetinginterestsweredisclosed.

    Grant informationTheauthor(s)declaredthatnograntswereinvolvedinsupportingthiswork.

    AcknowledgmentsWearethankfultoHarpreetSingh(TerritoryManager,SigmaAldrich,Gujarat,India)forprovidingthecustomsynthesizedtemplateDNAandoligonucleotideprimersusedinthisstudy.

    References1. WatsonJD,CrickFH:MolecularstructureofnucleicacidsastructureforDeoxyriboseNucleicAcid.Nature.

    1953171(4356):737738.PubMedAbstract|PublisherFullText

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

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

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

    5. TchurikovNA,ShchyolkinaAK,BorissovaOF,etal.:SouthernmolecularhybridizationexperimentswithparallelcomplementaryDNAprobes.FEBSLett.1992297(3):233236.PubMedAbstract|PublisherFullText

    6. BorisovaOF,ShchyolkinaAK,ChernovBK,etal.:RelativestabilityofATandGCpairsinparallelDNAduplexformedbyanaturalsequence.FEBSLett.1993322(3):3046.PubMedAbstract|PublisherFullText

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

    8. TchurikovNA,ShchyolkinaAK,BorissovaOF,etal.:SouthernmolecularhybridizationexperimentswithparallelcomplementaryDNAprobes.FEBSLett.199210:297(3):2336.PubMedAbstract|PublisherFullText

    9. MullisKB:Processforamplifyingnucleicacidsequences,UnitedStatesPatent4683202.1987.ReferenceSource

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

    11. BhardwajV,SharmaK:Dataset1.DNAsequencingfileforPCRandPDPCR.F1000Research.2014.DataSource

    12. BhardwajV,SharmaK:Dataset2.RealtimePCRfileforPCRandPDPCR.F1000Research.2014.DataSource

    OpenPeerReviewCurrentRefereeStatus:

    Version1

    RefereeReport19ene2015

    RamGopal,DepartmentofCellandMolecularBiology,UppsalaUniversity,Sweden

    Approved

    ItisinterestingtoreadthearticleParallelDNApolymersechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplatebyBhardwajandSharma.Theauthorsforthefirsttimehavedemonstratedthatprimerscanannealinparallel... Continuereading

    RespondorComment

    RefereeReport12ene2015

    HarishkumarMadhyastha,DepartmentofAppliedPhysiology,FacultyofMedicine,MiyazakiMedicalCollege,Japan

    Approved

    Thetitleoftheresearchpaperiswelljustifiedandsuitabletothecontentofthesubject.Abstractofthepaperiswellwrittenandconcluded.Materialsandmethod,dataanalysisanddesignoftheexperimentarecommendable.Inmyopinion,... Continuereading

    RespondorComment

    DiscussthisArticle

    Version1

    AuthorResponse03mar2015DrVikashBhardwaj,LovelyProfessionalUniversity,Punjab,India,India

    DearReinerVeitiaWehaveattachedtwoseparatecommunicationswereceivedearlierfromyou.ThefirstonesaysIhavereadyourpreprintonparallelDNAwithlotofinterest.It... Continuereading

  • 2/4/2015 ParallelDNApolymerasechainreaction:SynthesisoftwodifferentPCRproductsfromaDNAtemplateF1000Research

    http://f1000research.com/articles/3320/v1 6/6

    ReaderComment23feb2015ReinerVeitia,France

    IhavenowhadtheopportunitytorepeatthisexperimentwiththesametemplateandparallelprimersasdescribedintheMSandunfortunatelyfailedtoobtainanyamplification(at... Continuereading

    AuthorResponse09feb2015DrVikashBhardwaj,LovelyProfessionalUniversity,Punjab,India,India

    CheckoutthefollowingreportpublishedatGenomewebsitebyMadeleineJohnson

    "StudyClaimsPCRfromPrimersAnnealedinParallelOrientation"

    CompetingInterests:Nocompetinginterestsweredisclosed.

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dna polymerase

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