TwistAmp® DNA Amplification Kits

Assay Design Manual

Unwind DNA’s possibilities

TM

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Contents

1. Assay Considerations 31.1 Reaction temperature 31.2 Quantificationandamplificationonset 31.3 Preventingtemplatecross-contamination 41.4 Assayoptimisation 41.5 Slowerrateofamplificationandlongeramplicons 51.6 ReverseTranscriptaseuse 6

2. Primer design considerations 72.1 Primerlength 72.2 Primersequence 82.3 Amplificationproductlength 82.4 Primerselection 92.5 ‘Difficultamplicons’ 142.6 ’Primernoise’ 152.7 Degenerateprimersandmismatches 15

3. Probe design considerations 163.1.1 TwistAmp®exoprobestructureandfunction 173.1.2 TwistAmp®exoprobelength,positionandexamples 193.2.1 TwistAmp®nfoprobestructureandfunction 223.2.2 TwistAmp®nfoprobelength,positionandexamples 253.3.1 TwistAmp®fpgprobestructureandfunction 263.3.2 TwistAmp®fpgprobelength,positionandexamples 27

4. Multiplexing 294.1 Primercompatibilityandconcentrationratios 294.2 Inhibitoryprimers 294.3 Totalprimerconcentration 294.4 Differentamplificationrates 30

5. Sample input compatibility 305.1 Examplelysismethods 305.2 Buffercompatibility 305.3 Terminationofamplification 31

6. Other RPA methods 31

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Assay considerations1.1 Reaction temperatureThestandardTwistAmp®kitsareconfiguredtooperateoptimallyinthetemperaturerangeof37°Cto42°C.Athighertemperaturesthesystemwillbecompromisedastheenzymesprogressivelyloseactivity.Attemperaturesbelowtheoptimalrange,RecombinasePolymeraseAmplification(RPA)canworkexcellentlyalbeitatareducedreactionrate.However,thereagentcompositionandprotocolsofthecurrentTwistAmp®kitshavebeenoptimisedforveryfastamplificationandarenotspecificallyoptimisedforprotocolsusingtemperaturesbelowtherecommendedoptimalrange.Withreducedtemperatures,amplicondoublingtimeslengthenmorerapidlythanenergyconsumptionrates,whichcanleadtofuel‘burn-out’beforeaccumulation.SensitivitytotemperaturehasbeenmostacutelyobservedfortheTwistAmp® exo real-timefluorescencekits.TheotherkitsaresomewhatmoreresistanttolowertemperaturesandampliconshavebeensuccessfullygeneratedanddetectedattypicalambienttemperatureswithTwistAmp®BasicandTwistAmp®nfokits.

1.2 Quantification and amplification onsetTheonsettimeofdetectableamplificationforagivenassaywilldependontheamountofstartingtemplatematerial:themoretemplatecopiestherearetostartwith,theshorterthetimetodetection.However,exploitingthis‘time-based’quantification(asopposedtothecycle-basedqPCRapproach)demandsacarefulexperimentalsetup.Firstly,itiscriticaltoensurethesimultaneousinitiationofcomparedreactions,forinstanceofatestsampleversusastandarddilutionseries.Thiscanbeachievedthrough‘magnesiumacetatestart’asdescribedintheinstructionmanuals.Alternatively,reactionscanbetemporarilysloweddown(orhalted)bysettingthemuponiceandthenstartingbysimultaneoustransfertotheoptimalreactiontemperature.Slowing/alteringtheoverallkineticratetoimprovethetimeresolutionofassaysmayalsobeachievedbyalteringreactioncomposition,and/ortemperature.However,asstatedabove,thecurrentTwistAmp®kitshavenotbeenoptimisedforreducedkineticsandlowertemperatureandwedonotadviserunningtheTwistAmp®exosystembelow

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therecommendedtemperaturesaslittle,tonodetectablelevelsofamplificationmaybeobserved.

1.3 Preventing template cross-contaminationItisessentialtotakeprecautionstominimisethepotentialforcarryoverofnucleicacidmaterial(inparticularamplificationproduct)fromoneexperimenttothenext.Theuseofseparateworkareasandpipettorsforpre-andpost-amplificationstepsandtheuseofpositivedisplacementpipettorsoraerosol-resistantpipettetipsaregoodmeasurestotake,alongwiththeuseofPCRhoodswhereavailable.UsedpipettetipsandreactiontubesshouldbecollectedinairtightcontainersorunderconditionsthatdestroypotentiallycontaminatingDNA(e.g.acidicconditions,bleach,etc.).Extracarehastobetakenwithanytypeofpostamplificationprocessingofthereactionwherebytubescontainingampliconarerequiredtobeopened,suchaswhenconductingpurification,gelelectrophoresis,orlateralflowdetection.

1.4 Assay optimisationThestandardreactionconditionsdeliveredbythecurrentTwistAmp® kit formulationandprotocol(outlinedintheinstructionmanuals)allowbothfastandsensitiveamplification/detectionformostassays.However,theperformanceofsomeassayscanbeimprovedbyoptimisationofthereactionconditions.Parametersthatcaneasilybechangedbytheuserarethereactiontemperature,themagnesiumacetate(MgOAc)concentration,themixingregimeandtheprimer/probe(whereprobesareapplicable)concentrationsandratio:

•thetemperatureshouldbevariedwithintherecommendedrange(seeSection1.1).•therecommendedrangeofMgOAcconcentrationsinthereactionisbetween12mMand30mM(thestandardrecommendationoftheinstructionmanualsis14mM–reactionratecanincreasewithMgOAcconcentration).•thetimeofmixingcanbevariedbetween3minutesand7minutesafterinitiationofthereaction(standardtimeis4minutes–longerormoreslowlyaccumulatingampliconsmaybenefitfromslightlylater

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mixingalongwithRTassays.•theconcentrationofeachprimercouldbevariedbetweenapproximately150nMand600nMandtheconcentrationofprobecanbevariedbetween50nMto150nM.Inadditiontheratiosofprimer1:primer2:probecanbevaried,however,thetotaloligonucleotideconcentrationinthereactionshouldbemaintainedintherange750-2000nM.Usinglowconcentrationsofprimersmayreduceamplificationspeed,butbenefitlongerampliconsandimprovereal-timeresolution;whereashighconcentrationscanacceleratekinetics.

Note:TwistDxhasobservedbatch-to-batchvariationsinthequalityofprimerpreparationsfromcommercialsupplierswhichcanaffectRPAperformance.Forapplicationswhereconsistencyiscritical,wewouldrecommendtheuseofpurifiedoligonucleotidesinestablishedassays.Stockconcentrationsofprimersshouldalsobeverifiedbyappropriatemeansinrepeatordersassupplierconcentrationscanoftenbemisleading.

1.5 Slower rate of amplification and longer ampliconsThecurrentTwistAmp®kitformulationsaredesignedforthefastandsensitiveamplificationofrelativelyshorttargetsequenceswithintemplateDNA(80-500bp).Insomecasesotherparametersmaybedesired.Forexample,insomecircumstancesaslowerrateofamplificationmightbedesirable,suchaswhentheTwistAmp®processisgoingtobeusedfortemplatequantification(seeSection1.2).TherearemanywaystoengineerdecreasesinRPAreactionkinetics-forexamplesomeprimersjustamplifymoreslowlythanothersandmaybeselected.AlternativelythereactiontemperatureorMgOAcconcentrationcanbelowered.However,RPAreactionscontainalimited‘clock’dictatedbytheavailabilityofenergyregenerationcomponents,andwiththecurrentkitsarelativelyhighlevelofATP-burningrecombinase(selectedtopromoterapidkinetics)meansthatfuelisconsumedtypicallywithinabout25-30minutes(dependingonprimerconcentrationsused–thecomplexofDNAandrecombinasedictatesfuelconsumptionrate)regardlessofmodificationofreactionconditionswithinacceptable

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parameters.AsthereisonlyacertaindegreeofflexibilityintheratioofprimertorecombinasethatcanbeusedcurrentTwistAmp®kitsmaynotnecessarilybeperfectlysuitedforhighresolutioninreal-time.Thisisbecauseverylowamplificationrates(e.g.causedbyamplificationatlowtemperature)canleadtoATPordNTP-exhaustionpriortosignalgeneration(particularlywiththeTwistAmp®exokitsbecauseexonucleaseIIIintroducessomecompetitionwithpolymerasewithregardtoproductgeneration).TheTwistAmp®processmayalsobeemployedtogeneratelongeramplificationproducts,however,thecurrentTwistAmp®kitformulationsarenotspecificallydesignedforthispurpose.Nevertheless,itispossibletosomeextenttoimprovetheamplificationoflongerproducts,ortodeliberatelyslowamplificationforreal-timeanalysis,usingtheoptimisationstepsdescribedearlier(seeSection1.4,e.g.lowertheoligonucleotideconcentration,alterMgOAcandtemperaturewithinacceptablebounds).Weexpecttoreleasesomespecialisedkitsforlongerampliconsinthenearfuture.

1.6 Reverse Transcriptase useCurrentTwistAmp®RTkitscontainareversetranscriptasethatallowssingle-stepconversionofRNAintocDNAandsubsequentamplification.Noadditionalprimersarerequiredforthisprocesstooccur.It’s possible to use RNA template with a TwistAmp® kit just by adding asuitable reverse transcriptase (RT) when setting up a reaction. If an RTthat works at 37–42 °C is added to RPA chemistry then RNA can bereverse transcribed and the cDNA produced and amplified all in onestep. The complementary DNA sequence will be that of the RNA added.We recommend using similar RT amounts to that of a PCR reaction ofthe same volume, according to your chosen manufacturer’s instructions.We also suggest that you run reactions at 40°C and delay agitation ofthe reaction by a minute to give the RT time to work, otherwise you justrun the reaction as you would a normal TwistAmp® reaction (it’s a one step process). It’s also advisable to add RNase Inhibitor to any reactionwhere RNA is the target material.

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Primer design considerationsEstablishingasensitiveandrapidTwistAmp®assaydependsonselectingsuitableamplificationprimers.Sinceitisnotyetpossibletopredicttheamplificationperformanceofagivenoligonucleotidebasedpurelyonitssequence,itisrecommendedtoundertakeasimpleassaydevelopmentprocessincludingthedesignandthescreeningofaseriesofcandidateprimersandselectingapreferredprimerpair.

Note:Theprimerdesignguidancebelowisadvisableforhighsensitivityassaydevelopment.Forassaysrequiringlessstrigentdetection(greaterthan1000copiespersampletested)onlyasmallnumberofprimersneedbescreenedinmostcases,anduseofPCRprimersmaybesufficient.

2.1 Primer lengthPrimersdesignedforagivenPCRassaymayoftenworkinRPA,butmaynotbeoptimalforTwistAmp®reactions.TwistAmp®primersareideallylongerthantypicalPCRprimers,30to35nucleotideslong,andincontrasttoPCR,themeltingtemperatureofanoligonucleotideisnotthecriticalfactorforitsperformanceasaprimer.Theabilityofrecombinaseproteinstostimulateandcompleterecombination/primingdecreaseswitholigonucleotidesize.Conversely,whilemanyTwistAmp®primerscanalsobeusedinPCRamplification,theyarenotselectedoroptimisedforthatpurposeandtheirperformanceinPCRmaynotbearadirectrelationshiptotheirqualityasTwistAmp®primers.Oligonucleotidesshorterthan30nucleotidescanstillfunction,however,undertheseconditionstheamplificationkineticsaretypicallyslowercomparedtotheuseoftwoopposingamplificationprimersof30residuesorgreater.Oligonucleotidesofupto45nucleotideshavebeensuccessfullyusedasprimersintheTwistAmp®processandinprincipleprimerscouldbeevenlonger.However,lengtheningtheprimerdoesnotnecessarilyimprovetheamplificationperformanceanditincreasesthelikelihoodofsecondary-structuresthatcouldleadtoprimernoise(seeSection2.6).Itisthereforeadvisablenottodesignexcessivelylongprimers.

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2.2 Primer sequenceOligonucleotidesofdifferentsequenceperformdifferentlyinTwistAmp® reactions,buttherearenofixedrulestopredicthowwellagivenamplificationprimerwillworkbasedontheorderandcompositionofitsnucleotides.However,someguidelineshaveevolvedbasedonempiricalobservations(althoughtheseshouldnotdetertheuserfromgenerallytryingmanysequences).Wherepossible,itisbesttoavoidunusualsequenceelementswithintheprimer,suchaslongtracksofoneparticularnucleotideoralargenumberofsmallrepeats.Excessivelyhigh(>70%)orlow(<30%)GCcontentislikelytobedetrimental.Sincebase-pairinginteractionsbothwithinandbetweenprimerscouldcontributetoartefactgeneration(primerdimersetc.),oligonucleotidesthatcontainsequenceelementsthatpromotesecondarystructuresandprimer-primerinteractionsorhairpinsshouldbediscarded.

2.3 Amplification product lengthDependingonthereactionconditionsRPAallowsfortheamplificationofDNAproductsofuptoatleast1.5kb[http://dx.doi.org/10.1371/journal.pbio.0040204].However,thecurrentTwistAmp®kitshavebeenconfiguredtofavourrapidamplificationasopposedtomaximisingthelengthofamplificationproducts.Forthisreason,targetsofoverabout500bpdonotamplifywellwiththecurrentTwistAmp®formulation.Forultra-rapidTwistAmp®assayswerecommendanampliconlengththatdoesnotexceed500bp,andideallyisbetween100-200bp.Thisisbecauseshorterproductsaregeneratedinashorterperiodoftimeandthereforetendtohaveanimprovedproduct/noiseratio.Asaconsequence,theoverallamplificationperformanceisimproved(althoughamainfactorinamplificationspeedisacharacteristicofthechosenprimerpair).

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Note:AmpliconsizeinRPAdirectlyaffectstheamplificationsensitivityandspeed:duetotheeffectsofprimernoise(artefacts;seeSection2.6).Whilstlongerampliconsrequireincreasedtimetoamplify,primernoisetendstobeindependentoftargetsize.Therefore,thelongeranampliconis,themorelikelyprimernoisewilloutcompetetargetamplification.Forthisreason,shortampliconsarepreferredforultra-sensitiveassays.

Ifprobesaretobeusedfordetection,thencaremustbetakenwhendefiningamplificationprimerstoleaveenoughsequencespaceforthedesignofanoligonucleotideprobe(seeSection3).ThelowerlimittothesizeofRPAproductsismainlydeterminedbythesizeofRPAprimers.Typicallythisrequiresthatampliconswillbelongerthanabout70-80bp.

2.4 Primer selectionTheprimerselectionprocesstypicallyconsistsofthefollowingsteps:

Step 1. Choice of target regionItisadvisabletoselectaregionwithinthetemplatethatischaracterisedbyrelatively‘average’nucleotidesequencecomposition:

•GCcontentofbetween40%and60%•repetitivesequences•fewdirect/invertedrepeats,palindromes,etc.

Repetitiveelementswithinagivengenomeshouldbeavoidedinordertopreservetheuniquenessofthetarget.InthisrespectthepreferredsequencesareidentifiedinmuchthesamewayasthoseforPCR.

Step 2. Primer candidatesAfterchoosingasuitabletargetregion,twogroupsofstaggeredoligonucleotidesfacingeachother(i.e.havingforwardandreversedirection)areselectedthatserveasprimercandidates(seeFigure2).Primersinthesameorientationcan,butneednot,beoverlapping.Eachprimerfromtheforwardgroupcanlaterbepairedwitheachprimerfrom

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thereversegroup.AmediumscalescreendesignedtodetecttemplateDNAwithsinglemoleculesensitivitywithfasterthan20second‘doublingtimes’(thelengthofreactiontimeduringtheexponentialphaseofRPAinwhichtheamountofproductdoubles)typicallyhas8to10primersperdirection(i.e.creating64to100possibleprimercombinations).Whenunderstricttimeconstraintsandwhereoptimalprimerdesignisnotessential(suchasforanassaytargetfoundinrelativelyhighconcentrationinsamples),thefollowingvalueinputsaresuggestedasPCRsoftwaresettings(e.g.Primer3,Primer-BLAST):

•primersizeminimumof30andmaximum36•primerGC%minimumof20andmaximum70•primerTmminimum50andmaximum100•maximumallowablelengthofamononucleotiderepeat,e.g.‘CCCCC’setto5.

Step 3. Screening of candidatesOncecandidateprimerpairsaredefined,theirrelativeperformanceshavetobeassessedandcompared.Thequalityofeachprimerpairdependsonthecontextoftheintendedassay,andforthisreasonthemethodofread-outforthescreeningprocedurediffersaccordingly:

•forTwistAmp®Basiceachprimerpairshouldbeusedasdescribedintheprotocol,thevariousamplificationproductsshouldbepurified(e.g.standardPCR-productpurificationmethods)andthenresolvedbyagarose-gel-electrophoresis.Thevariousprimerpairscanthenbeclassifiedaccordingtosensitivity,productyield,product/noiseratioandamplificationtime(ifatimecourseistaken).•forTwistAmp®exoandTwistAmp®fpgtheprimerpairsaretestedusingtherelevantfluorescencedetectionprobesaccordingtothestandardprotocolforareal-timeread-outandthefluorescencedataiscompared.Thekeyperformanceparametersaresensitivity,timeofamplificationonsetandtotalfluorescencesignalstrength.•forTwistAmp®nfoprimerpairqualityisassessedusingTwistAmp®

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nfoprobesandthecorrespondinglateral-flowstripanalysisasread-outsystem.Suitableperformanceindicatorsaretotalsignalstrengthandabsenceofbackgroundsignalonthelateral-flowstrip,aswellassensitivityandamplificationtime(ifatimecourseistaken).

Itshouldbenotedthatinpracticethemostpracticalassaymethodforprimerscreeningisprobablybyuseofareal-timefluorescenceread-outwhichrapidlyprovidesbothsensitivityandkineticdata.Primerpairsandprobescanthenbeadaptedforappropriatereadouti.e.nfoprobe,Basic.Requalificationmaybeperformedtoconfirmchoice.Itisnotalwaysnecessarytotesteverypossibleprimercombination.Forexample,byscreeningallreverseprimersagainstasingleforwardprimer,pickingthebestreverseprimerandthenusingittoscreenalltheforwardprimers,agoodprimerpaircanbefoundin16to20reactions(seeFigure1aand1b).

Figure 1a. Example of a 5x5 primer candidate matrix and the results of a first screening experiment. All reverse primers (designated R1 to R5) are screened with forward primer 3 (F3) and scored for amplification performance (‘-‘ for failed amplification, ‘+++’ for best amplification, etc.). Here, reverse primer 4 (R4) gives the best results.

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Figure 1b. Example of a second screening experiment based on the result shown in figure 1a. R4 is screened with all forward primers. F4 gives the best results. The combination F4/R4 is the best performing primer pair. Further screening experiments can be performed to confirm this finding, e.g. screening all the reverse primers against F4.

Thetestconditionsforthecandidatescreen(copynumberoftemplate,templatepurity)shouldbesufficientlystringenttomakeitpossibletodistinguishprimerqualityatthetopendoftheperformancelist.Ontheotherhand,initialconditionsshouldnotbesochallengingthatnoneoftheprimerpairstestedissuccessfulatthisstageoftheassaydevelopmentprocess:evenifthegoalistodetectonemolecule,findingthelimitedsetofprimersthatdetect25moleculeswillnarrowthefieldtothosethataregenerallywell-behavedandcanpotentiallybeimproved(seeStep4).InmanycasesStep3ofthescreeningstrategywillalreadyyieldsufficientlygoodprimerpairsfortheintendedassay.Ifso,thebestprimerpairs(typicallythebest3)canthenbefurthercharacterisedforamplificationspeedandsensitivitybyrepeatedtestingatdifferentstartingtemplateconcentrations.Itisadvisabletodecideonashort-listofsuitableprimerpairsatthisstage(ratherthanjustonepair)ifmultiplexingisrequired(seeSection4).Ifnoneoftheprimerpairstestedatthisstageoftheassaydevelopmentprocessperformssatisfactorily,itisnecessarytoreturntoStep2anddesignnewprimers.

F1

R1 R2 R3 R4 --

R5

F2 ++ F3 -- + + +++ ++ F4 ++++ F5 ++

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Note:Thesuccessofaprimerscreensetupcanalsodependonthequalityoftheprobe;seeSection3foradiscussionofdetectionprobedesign.

Step 4. Secondary and tertiary candidate screen (for high sensitivity assays)Evensmallvariationsinthesequenceofoligonucleotidescansometimesresultinsignificantdifferencesinprimeractivity(possiblyduetoadifferentpropensitytoformprimerdimersorotherartefacts).AstrategytoimprovetheperformanceofagivenassayisthereforetogenerateasecondgenerationofprimersbycreatingvariantsofthebestprimersidentifiedinStep3andre-screentheseforimprovedamplificationperformance.Agoodfirststepistofillinthegapsaroundtheprimersselectedintheinitialscreen.Screenprimersofthesamelengthasthoseselected,butmovedin1baseincrementsaroundthemuptorejectedprimers.Furtherrefinementscanbemadebyaddingandsubtractingbasesfromthe3’endofthechosenprimers(sometimesa32-merwillperformbetterthana35-mer,sometimesa38-merwillperformbest).Step4oftheprimerselectionprocedureshouldyieldgoodprimerpairsfortheintendedassay.Ifthisisnotthecaseandnoneoftheprimerpairstestedduringthedevelopmentprocessperformssatisfactorily,itisnecessarytoreturntoStep2anddesignnewprimers.

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Figure 2. Typical stringent primer screen. Where single molecule sensitivity is not required, the primary screen will usually suffice.

2.5 Difficult ampliconsOnrareoccasionssequenceelementswithinanamplicon(i.e.thesequencesbetweenprimers)aredetrimentaltorapidamplificationandallprimerpairsproducingampliconssharingsuchasequencemotifwillperformpoorly.Shouldattemptstodesignsuitableprimerpairsforagiventargetregionconsistentlyyieldresultsoutsideofthenormalrangeofexpectedordesiredbehaviour,itmaybeworthseekinganother,non-overlappingtargetregionelsewhereinthetemplate.The

Primary Screen: Primers flanking target sequence are tested in different combinations

Secondary Screen: Primers differing by 1 base increments around the best primary screen primers are tested in different combinations

Tertiary Screen: Primers differing in length by 1 base increments at the 3’ end of the best secondary screen primers are tested in different combinations

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normalexpectationforafirst-roundscreenwouldbetodetectaslowas25-50copiesandachievedetectionthresholdinreal-timewithin15minutesofattainingoptimalreactiontemperatureusingmoststandardfluorescencemonitoringsystems.

2.6 Primer noiseInadditiontopromotinggenuineamplificationevents,theTwistAmp® reactionenvironmentalsoallowsundesiredprimerinteractionstooccur(similarphenomenaarecommontoothernucleicacidamplificationtechniques,forexamplePCR).Suchinteractionscanbeintramolecular(hairpins,etc.)orresultfromprimerdimerformation,bothbetweenidenticalordifferentoligonucleotides.ThesestructurescanprovidesubstratesforextensionbyDNApolymeraseandsomeoftheartefactssogeneratedwillserveastemplateforfurtherrecombination/extensioneventsandthusenteraphaseofexponentialamplification.ProcessesofthistypewillgeneratedetectablelevelsofrelativelylowmolecularweightDNAconsistingofprimerderivedsequence(‘primernoise’).Noisereactionsareincompetition(forprimers,nucleotides,polymerasebindingandenergy)withthegenuineamplificationprocess,andwilleventuallyinhibitthelatter.Thepropensitytogeneratenoisewilllimitthesensitivityofagivenprimerpair.Forthisreason,itisimportanttoselectprimers,whichwillminimisethecompetitiveproductionofprimerartefacts.Inpracticethisrequiresscreeningofcandidateprimersforthosewiththehighestsensitivity.PrimernoisewillbeunrelatedinsequencetothatofthedetectionprobesusedwiththeTwistAmp®exo,TwistAmp®fpgandTwistAmp®nfokits.Suchartefactswillthereforenotgenerateanerroneoussignalwiththesekits.

2.7 Degenerate primers and mismatchesTherearenospecificsuggestedlimitsonuseofdegeneratebaseswithRPAotherthanminimaluseisprobablyadvisable.RPAisgenerallyquiterobusttomismatches.IthasbeenshownthattheperformanceofRPAassaysisnotaffectedwhenprimersandprobecarry5–9basepairmismatches(http://dx.doi.org/10.1371/journal.pone.0071642;http://dx.doi.org/10.1128/mBio.00135-13).

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Probe design considerationsRPAutilisesproprietaryprobesystemsdesignedforprovidingspecificandsensitivedetectionofamplicons.Theseprobesaretypicallydesignedtohavehomologytoregionswithinanampliconbetweenthemainamplificationprimers.OligonucleotideprobesthatarecompatiblewiththeTwistAmp®technologycomeinthreedifferentvarieties:TwistAmp® exo,TwistAmp®fpgandTwistAmp®nfoprobes.EachtypeisusedwiththeTwistAmp®exo,theTwistAmp®fpgkit,andtheTwistAmp®nfokit,respectively.

Probe orderingTwistAmp®probescanbeorderedfromvariousoligonucleotidemanufacturers,usingtheTwistDx™TwistAmp®probeorderforms(availableattwistdx.co.uk).

Fluorophore choiceArangeoffluorophoresofdifferentwavelengthareapplicableforuse.FluorophorelabelspreviouslyusedincludeFAM,SIMA/HEX,ROX,TAMRA,TexasRed,andCalFluor610.WhenusingFAMfluorophoreitisrecommendedtouse6FAM,astheemissionbandfromthe6-carboxyfluoresceinoligonucleotidehasbeenshowntobesubstantiallysharperthanthatofthe5-carboxyfluoresceinanalogue(https://doi.org/10.1021/bc7001874).Whenusingbiologicalsamples,itisrecommendedtoavoidFAMuseasthesesamplesmayexhibitahighlevelofbackgroundfluorescence.Suggestedsuitablefluorophorequenchersarelistedbelow:

Fluorophore QuencherFAM BHQ1SIMA/HEX BHQ1ROX BHQ2TAMRA BHQ2TexasRed/CalFluor610 BHQ2

ThestructuresofallthreetypesofTwistAmp®probearediscussedseparatelybelow.

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Probe candidatesAlthoughprobesappeartobelesssensitivetosequencevariationsthanprimers,probesofdifferentsequencewillperformdifferently.Ifoptimalperformanceisrequireditisadvisabletotestmorethanonepotentialprobewithinatarget.However,evenwithoutoptimisationmostprobesdesignedaccordingtothedescribedprinciplewillworkadequatelyandbesuitableforthepurposeofdistinguishingtheperformanceofdifferentprimerpairs.Ifaprobeistobeusedforaprimerscreen,itisagoodstrategytodesignitsothatitislocatedwithinthesmallestcandidateamplicon(definedbytheinnermostprimersintheforwardandreversegroupsofcandidateprimers,seeSection2).Theprobecanthenbeusedtotesttheperformanceofallthesurroundingprimers.Itisworthnotingthatprobescanbedesignedforeitherstrandwhichincreasesthenumberofpossiblecandidatesthatcanbedesignedforagiven target.

3.1.1 TwistAmp® exo probe structure and functionThetypeofoligonucleotideprobethatiscompatiblewiththeTwistAmp® exokitistheTwistAmp®exoprobe.TwistAmp®exoprobeconsistofanoligonucleotidebackbonethatcontains:

•anabasicnucleotideanalogue(atetrahydrofuran(THF)residue,sometimesreferredtoasa‘dSpacer’).•aflankingdT-fluorophore(typicallyfluorescein,butanyfluorophoreavailableasdT-coupledreagentsforoligonucleotidesynthesiscanbeused).•acorrespondingdT-quenchergroup(typicallyasuitableBlackHoleQuencher(BHQ))ontheothersideoftheTHFgroup.•asuitable3’–modificationgroup(suchasaC3-spacer,aphosphate,abiotin-TEGoranamine)toblocktheprobefrompolymeraseextension.

Anyfluorescentsignalgeneratedbythefluorophorewillnormallybequenchedbythequencherlocated2-5bases3’tothefluorophore.InadoublestrandedcontexttheTHFresiduepresentsasubstratefortheDNArepairenzymeExonucleaseIIIpresentintheTwistAmp® exo

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kit,whichwillcleavetheprobeattheTHFposition,therebyseparatingthefluorophoreandthequencherandgeneratingafluorescentsignal.Thisnucleaseactivityisrestrictedtowhentheprobehasannealedtothetargetsequencewithintheamplificationproduct.Thecuttingoftheprobeisthereforeindicativeofthespecifictargetamplificationeventandcanbeusedtomonitorspecificampliconaccumulation.AstheinternallabelsusedinTwistAmp®exoprobearecurrentlyonlyreadilyavailableonthymines,thereisgenerallyarestrictionofidealprobelocationstosequencesinwhichtwothyminescanbefoundwithfewerthanabout5interveningnucleotides(greaterseparationsdecreasethequenchingefficiencyandatpresentnucleotideanaloguesforotherbasesarenotavailable).Howeverinmostcases,andgiventhateitherstrandofthetargetcanbeused,thereareusuallyappropriatedesignsitesavailable.Furthermore,therearetwoadditionalapproachesthatmaybeusedtoexpandtheavailablesitesforTwistAmp® exo probe design.(1)TheTwistAmp®exoprobecanbeidenticaltooneoftheamplificationprimersbutpossessanadditional3’sequenceextensioncontainingtheTHFresidueandthefurther15residuesofhomology3’totheTHFresidue.TheTHFresiduemustbepositionedfurther3’tothesequenceoftherelatedmainamplificationprimertoavoidtheprobedetecting‘primernoise’eventsthatmaybegeneratedfromit.(2)Itisalsopossibletotolerateamismatchintheprobesuchthatiftwoconvenientlyseparatedthyminescannotbelocatedonecansimplyignorethemismatchofoneofthethyminesintheprobewiththetargetsequence.Theremaybeareductionintheefficiencyoftheprobe,theextenttowhichcannotbepredicted;howeversuchconfigurationshavebeeneffectiveinthepast.Figure3showsaschematicofatypicalTwistAmp® exo probe.

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TwistAmp® exo probeExonuclease III cuts THF residue

exo

5’ 3’

30+ bases 15 bases

fluorophore

3’ block

exo

THF residue

quencher

Figure 3 Schematic of the structure of an annealed TwistAmp® exo probe. The abasic THF residue is cleaved by Exonuclease III only when the probe is bound to its target. This cutting step separates the fluorophore and quencher and generates a fluorescent signal.

3.1.2 TwistAmp® exo probe length, position and examplesATwistAmp®exoprobeshouldbe46-52nucleotideslong,atleast30ofwhichareplaced5’totheTHFsite,andatleastafurther15arelocated3’toit.

Note:TheTHFresidue,thedT-fluorophoreanddT-quencherreplace basesfoundwithinthetargetampliconsequenceandarenot additional insertions.

Thereisnofixedruledescribingthebestpositionofagivenproberelativetoitscorrespondingamplificationprimers.Caremustbetakentoavoidthepossibilitythatprimerartefactscanbedetectedbytheprobe,ascouldoccuriftheprobeoverlapstheamplificationprimers.Primersopposingthedirectionoftheprobeshouldnotoverlaptoavoidtheoccurrenceofprimer-probedimers.However,primersinthesamedirectionastheprobecanoverlapits5’part(seeSection3.1.1)thisoverlapmustnotincludetheabasic-siteandmore3’partsoftheprobe(i.e.theoverlapoftheprimershouldberestrictedtothe5’-most27-30nucleotidesoftheprobe).Thiswillpreventtheinadvertentgenerationofartefactualhybridisationtargetsforthesensitivecleavagesequenceelementoftheprobe.Secondarystructuresthatcouldcauseprobesto

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foldbackonthemselvesshouldalsobeavoided.

Example TwistAmp® exo probe designsGivenanappropriatetargetsequencethemostimportantfactoristoidentifyapairofTresiduesincloseproximitytooneanother(withonly1-5interveningnucleotides).Asanexampleatargetsequenceisshownbelowalongwithtwosuggestedprobesthatcouldbedesignedtodetectit:

ATAGAGATCATCTCGAGATCGACGACTCGAGGGAATTTCAGAGGCTATA

GCGATCTCAGGTACATCGATAGATCGCTAGATAGCTCGCTAAACTCTCG

GACTCATCTAGCTCGATCGGATAATCGATATCGATATAGGCGGCGGCTA

5’

3’

fluorophore

THF residue

3’ block

quencher

Figure 4 An example of a sequence and possible TwistAmp® exo probes that could be generated for this sequence. The positions of the possible probes are indicated with a bold line, probe attachments and structures are labelled accordingly. Corresponding bases to be replaced are indicated in red.

InthiscaseoneprobeorderedwouldhavethefollowingsequenceinwhichtherelevantTresiduesinthesequencearereplacedbydT-fluorophoreresiduesordT-quencherresidues,andonebase(aCinthiscase)isreplacedbytheTHFresidue:

GAATTTCAGAGGCTATAGCGATCTCAGG[FAM-dT]A[THF]A[BHQ1-dT]CGATAGATCGCTA[3’-block]

ThenumberofnucleotidesbetweenthedT-fluorophore,orthedT-quencher,andtheTHFcanbe0,1or2andthereisnoknownsequencerequirementfortheseinterveningnucleotides,norforthebasewhichisreplacedwithaTHF.Basedontheseprinciplesasecondpossibleprobeisshownwithsequence:

TCGGACTCATCTAGCTCGATCGGATAA[FAM-dT]CG[THF]TA[BHQ1-dT]CGATATAGGCGG[3’-block]

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Wehaveroutinelyblockedthe3’-endoftheprobewithagroupsuchasC3-spacer,biotin-TEG,orphosphate.Amplificationprimersaredesigned,inmostcases,toflanktheprobesequences,howevertherecanbesomeoverlapbetweenthe5’portionoftheprobeandanamplificationprimerasdetailedabove.

Common TwistAmp® exo probe design errors:Targetsequenceforprobe:

GAATTTCAGAGGCTATAGCGATCTCAGGTCAATCGATAGATCGCTA

F=FAM-dT H=Tetrahydrofuran Q=BHQ1-dT

Good probe design: GAATTTCAGAGGCTATAGCGATCTCAGGFAHAQCGATAGATCGCTA[3’-block]≥30bases5’ofTHF≥15bases3’ofTHF≤5basesbetweenfluorophoreandquencherSingleTHF,betweenfluorophoreandquencher

Poor probe designs: GAATTTCAGAGGCTATAGCGATCTCAGGFHHHQCGATAGATCGCTA[3’-block]OnlyoneTHFneededperprobe.

GAATTTCAGAGGCTATAGCGATCTCAGGTACATCGAFAHAQCGCTA[3’-block]Thereshouldbe≥15bases3’oftheTHFiftheexonucleaseisgoingtocutitefficiently.

GAATTTCAGAGGCTATAGCGATCFCAHGQACATCGATAGATCGCTA[3’-block]Thereshouldbe30-38bases5’oftheTHFsothattheprobecanactasaprimerwhenitiscut.

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GAATTTCAGAGGCTATAGCGATCTCAGGFAHATCGAQAGATCGCTA[3’-block]Distancebetweenfluorophoreandquencheristoolarge–quenchingmaybepoor.

GAATTTCAGAGGCTATAGCGATCTCAGGFACHAQCGATAGATCGCTA[3’-block]THFhasbeenaddedasanadditionalbase,ratherthanreplacinganexistingbase.

3.2.1 TwistAmp® nfo probe structure and functionTwistAmp®nfoprobesareusedwiththeTwistAmp®nfokit,andareintendedfordetectionbyso-calledsandwichassays,onlateralflowconsumables.Theprobesconsistofanoligonucleotidebackbonewith:

•a5’-antigeniclabel.•aninternalabasicnucleotideanalogue(atetrahydrofuranresidueorTHF–sometimesreferredtoasa‘dSpacer’)whichreplacesanucleotide.•apolymeraseextensionblockinggroup(suchasaphosphate,C3-spaceroradideoxynucleotide,butnot a biotin-TEG)atthe3’end.

TwistAmp®nfoprobesareusedinaconfigurationinwhichtheopposingamplificationprimerislabelledatits5’endwithanotherantigeniclabel(differenttothatoftheprobe).Thethirdoligonucleotidepresentinthereaction(equidirectionalwiththeprobe)isaconventionalprimer.Figure5showsaschematicofatypicalTwistAmp®nfoprobe.Inpractise,itmakesnodifferencewhichwayroundthelabelsareusedi.e.whichlabelisontheprobe,andwhichoneisontheprimer.However,foreaseoffurthertestdevelopment,itmayproveusefultoputthelabelfortheconjugateantibodyontheprobe(BiotinforPCRD).Thisway,userswhoproceedtouseatwolinestripformultiplexing,canchangewhichlineonthestriptheydetectatbyorderinganewlabelledprimerratherthanorderinganewprobe.

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Note:TheTHFresiduereplace a basethatwouldbepresentinthetargetsequenceandisnot an additional insertion.

Figure 5. Schematic of the structure of an annealed TwistAmp® nfo probe. The abasic THF residue is cleaved by nfo only when the probe is bound. The 3’ OH group generated in the process is a target for extension by the polymerase and enables the 5’ label to be integrated in amplification products.

TheTwistAmp®amplificationreactionpromotedbythetwoprimeroligonucleotideswillgeneratetargetsfortheannealingoftheTwistAmp® nfoprobe.IntheresultingdoublestrandcontexttheTHFresiduepresentsasubstratefortheenzymenfo(alsoknownasEndonucleaseIV)presentintheTwistAmp®nfokit.nfowillcleavetheprobeattheTHFpositionandtherebygenerateanew3’-hydroxylgroup(effectivelyde-blockingtheprobe)thatcanactasaprimingsiteforpolymeraseextension,thustransformingtheprobeintoaprimer.Theampliconproducedbytheprocessedprobeandthe5’-labelledopposingamplificationprimerwilleffectivelyco-jointhetwoantigenicresiduesinoneDNAmolecule(seeFigure6).Thisduplexcanthenbedetectedinsandwichassayformats(typicallypost-amplification,i.e.endpointdetection),usingconsumablessuchasthoselistedbelow:

Consumable Designed to detect amplicons labelled withMileniaHybridetect-1 biotin+FITC/FAMU-Starunits biotin+FITC/FAMPCRD DIG+biotinand/orFITC/FAM+biotin

TwistAmp® nfo probenfo cuts THF residue nfo

5’ 3’

30+ bases 15 bases

5’ label

3’ block

nfo

THF residue

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Furtherinformationabouttheaboveconsumablescanbefoundattwistdx.co.uk.Lateral-flowstripsforthedetectionofnucleicacids(includingtheMileniaHybridetect-1)aretypicallydesignedtoutiliseproducts(forinstanceofPCRproductsandantigen-labelledprobes)astheirsubstrateandthereforerequireextensivesampleprocessingprocedures.IncontrasttheTwistAmp®nforeactionmechanismgeneratesthedouble-labelledreportermoleculesimultaneouslywiththeamplificationreactionandonlyrequiresminimalpost-amplificationprocessing.

B Co-joining of the two antigenic labels in an amplicon

anti-label A

anti-label B

label A

5’ label B

3’ block

nfo

THF residue

A TwistAmp® nfo probe hybridisation, cutting event, polymerase extension

5’ 3’

3’ 5’reverse primer

TwistAmp® nfo probe

gold particle

lateral flow strip

pol extension

Figure 6. Schematic of the arrangements of amplification primers and TwistAmp® nfo probe. (A) The processed probe and the opposing primer will generate double- stranded amplification products that co-join the two antigenic labels. (B) This product can subsequently be captured using one of the labels (for instance using the anti- biotin test line on a lateral flow strip), and visualised using the other (for example by interaction with gold-labeled antibodies).

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Thenfonucleasereactionandtheresultinggenerationofthedouble-labelledampliconarerestrictedtocasesinwhichtheprobecanannealtoitstargetsequence,chosentobewithintheoriginalamplificationproduct.CuttingoftheprobeisthereforeindicativeoftheamplificationeventitselfandcanbeusedtomonitorthesuccessoftheTwistAmp® reaction.

3.2.2 TwistAmp® nfo probe length, position and examplesATwistAmp®nfoprobeshouldtypicallybe46-52nucleotideslong,atleast30ofwhichareplaced5’totheTHF(tetrahydrofuran)siteandatleastafurther15arelocated3’toit.TheTHFresiduereplacesanucleotidethatwouldnormallybasepairtothecomplementarysequence.Thereisnofixedruledescribingthebestpositionofagivenproberelativetoitscorrespondingamplificationprimers.Caremustbetakentoavoidthepossibilitythatprimerartefactscanbedetectedbytheprobe.Althoughprimersthathavethesamedirectionastheprobecanevenoverlapits5’part,thisoverlapmustnotextenduptotheabasic-siteportionoftheprobe(i.e.theoverlapoftheprimershouldberestrictedtothe5’-most30nucleotidesoftheprobeorso).Thiswillpreventtheinadvertentgenerationofhybridisationtargetsforthesensitivesequenceelementoftheprobebyprimerartefacts.Primersopposingthedirectionoftheprobeshouldnotoverlaptoavoidtheoccurrenceofprimer-probedimers.Theopposingamplificationprimerhastobelabelledwithanantigenicgroup,usuallyabiotin.

Example TwistAmp® nfo probe designAsanexampleatargetsequenceisshownbelowalongwithasuggestedprobethatcouldbedesignedtoit:

ATAGAGATCATCTCGAGATCGACGACTCGAGGGAATTTCAGAGGCTATA

GCGATCTCAGGTACATCGATAGATCGCTAGATAGCTCGCTAAACTCTCG

GACTCATCTAGCTCGATCGGATAATCGATATCGATATAGGCGGCGGCTA

5’

3’

5’ label

THF residue

3’ block

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Figure 7. An example of a sequence and possible TwistAmp® nfo probe that could be generated for this sequence. The position of the possible probe is indicated with a bold line, probe attachments and structures are labelled accordingly. Corresponding base to be replaced is indicated in red.

Theprobeisblocked(typicallywithadideoxy-C,G,AorT,orwithaC3-spacer,oraphosphate,but not a biotin).Thesequenceofthisprobewouldthusbe:

GAATTTCAGAGGCTATAGCGATCTCAGGTA[THF]ATCGATAGATCGCTA[3’block]

AmplificationprimerswouldnormallyflanktheprobeoneithersideasindicatedinFigure6.

3.3.1 TwistAmp® fpg probe structure and functionTwistAmp®fpgprobeisusedwiththeTwistAmp®fpgkit,andisintendedforfluorescencedetectionassays.Theseprobestypicallyareoligonucleotideshomologoustothetargetampliconthatcontain:

•afluorophorelabelreplacinganAorGnucleotidewithadR-fluorophore(typicallyFAMorROX)positionedabout15nucleotidesupstreamfromthe3’endoftheprobe.ThefluorophoreisattachedtothedeoxyriboseoftheabasicsiteviaaC-O-Clinker(aso-calleddR-group).•adT-quencher(specifictothechosenfluorophoretype)isthenpositioned1to3nucleotideseitherupstream,ordownstreamofthefluorophore.•Inaddition,TwistAmp®fpgprobesareblockedfrompolymeraseextensionbyasuitable3’modification(suchasaC3-spacer,aphosphate,aBiotin-TEGoranamine).

Thefluorescentsignalgeneratedbythefluorophore(anyfluorophoresthatareavailableasdR-derivativessuchasCarboxy-fluorescein)willnormallybequenchedbythequenchergroup(typicallyaBlackHoleQuencher(BHQ)).Inadouble-strandedcontextthedR-fluorophore

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residue,the‘gap’intheprobe,presentsasubstratefortheenzymefpgpresentintheTwistAmp®fpgkit.fpgwillcleavetheprobeatthedRposition,therebyseparatingthefluorophoreandthequencherandgeneratingafluorescentsignal.Thisnucleasestepisrestrictedtocasesinwhichtheprobecanannealtoitstargetsequence,chosentobewithintheamplificationproduct.CuttingoftheprobeisthereforeindicativeoftheamplificationeventitselfandcanbeusedtomonitortheprogressoftheTwistAmp®reaction.Figure8showsaschematicofatypicalTwistAmp®fpgprobe.

Note: thedR-fluorophoreanddT-quencherreplace basesfoundwithinthetargetampliconsequenceandarenot additional insertions.

TwistAmp® fpg probefpg cuts dR group

fpg5’ 3’

15 bases

fluorophore

3’ block

fpg

dR group

quencher

Figure 8. Schematic of the structure of an annealed TwistAmp® fpg probe. The abasic dR residue is cleaved by fpg only when the probe is bound to its target. This cutting step releases the fluorophore from the probe and the quencher proximity and generates fluorescence signal.

3.3.2 TwistAmp® fpg probe length, position and examplesATwistAmp®fpgprobeshouldtypicallybe46-52nucleotideslong,butshorterprobesmayalsobesuccessful.ThereisnofixedruledescribingthebestpositionofagivenTwistAmp®fpgprobequencherrelativetotheamplificationprimerswithwhichitisused.However,caremustbetakentoavoidthepossibilitythatprimerartefactscanbedetectedbytheprobe,soanyoverlapbetweenprimersandtheprobeshouldbeavoided.

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Example TwistAmp® fpg probe designAsanexampleatargetsequenceisshownbelowalongwithtwosuggestedprobesthatcouldbedesignedtoit:

ATAGAGATCATCTCGAGATCGACGACTCGAGGGAATTTCAGAGGCTATA

GCGATCTCAGGTACATCGATAGATCGCTAGATAGCTCGCTAAACTCTCG

GACTCATCTAGCTCGATCGGATAATCGATATCGATATAGGCGGCGGCTA

5’

3’

dR-fluorophore

3’ block

quencher

Figure 9. An example of a sequence and possible TwistAmp® fpg probes that could be generated for this sequence. The positions of the probes are indicated with bold lines, probe attachments are labelled accordingly. Corresponding bases to be replaced are indicated in red.

InthiscaseoneprobeorderedwouldhavethefollowingsequenceinwhichafluorophorelabelattachedviaadR-groupispositioned15basesfromthe3’probeend,withadT-quencherpositioned2nucleotidesdownstreamofthefluorophore,andblockedwitha3’C3-spacer:

GAATTTCAGAGGCTATAGCGATCTCAGGT[dR-FAM]CA[BHQ1-dT]CGATAGATCGCT[3’-block]

BasedontheseprinciplesasecondpossibleprobeisshownwithdR-fluorophorepositioned16basesfromthe3’probeend,withadT-quencherpositioned1nucleotideupstreamofthefluorophore,andblockedwitha3’C3-spacer:

TCGGACTCATCTAGCTCGATCGGATAA[BHQ1-dT]C[dR-FAM]ATATCGATATAGGCGG[3’-block]

TheexactnumberofbasesbetweentheinternaldT-quencherandthedR-fluorophoreisadvisedtobekepttoamaximumof3basestomaintainthequenchingefficiency.

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MultiplexingAmultiplexTwistAmp®reactionisthesimultaneousamplificationofmorethanonetargetinasinglereaction.Theprimersusedfortheamplificationofeachofthemultiplexedtargetshavetobetestedandpotentiallyoptimisedforcompatibility.Notallprimerpairsthatprovetobesufficientlyactiveinsingleassayreactionswillturnouttoperformwellinmultiplexassayreactionsetups.

4.1 Primer compatibility and concentration ratiosAgoodstrategyforidentifyinggoodprimersformultiplexreactionsistofirstdefineanumberofgoodcandidateprimerpairsforeachtargetindividually.Subsequently,thecandidatepairsforeachtargetaresuccessivelycombinedwiththecandidatepairsoftheothertargetsinthefinalmultiplexformat.Afterhavingdefinedthebestcombination,differencesintherelativeperformanceoftheprimerpairsforthevarioustargetscanbeadjustedbychangingtheratiosoftheamountofprimersusedinthereaction(forinstance,inahypotheticalduplexreactiontheproportionofprimersusedtoamplifytargetAmightaccountfor65%ofthetotalprimeramountinthereaction,whiletheproportionofthoseusedfortargetBwillonlybe35%).

4.2 Inhibitory primersInsomecasesofmultiplexingitcanappeardifficulttopaircertainprimerpairseffectivelytogetherevenwhentheratiosofprimersareadjusted.Oftentimesthisisbecauseoneprimerinthesystemhasadominatingand/orinhibitingactivityonotherprimersandampliconsinthesystem.Inthiscaseitcanbeworthwhiletoaddevenonlyoneprimerofapairintoanotherwiseeffectivesystemtodeterminewhichsingleprimerunderanalysishasthisundesirableactivity.Oncetheprimerhasbeenidentifiedapproachesmaybetakentoexcludeitfromfuturemultiplexingexperiments.

4.3 Total primer concentrationItshouldbenotedthatthetotalamountofoligonucleotidesinthereaction(i.e.thesumofallprimersaddedtothereactionmixture)

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shouldnotexceedtherecommendedamount.Multiplexingwillthereforeleadtoareductionoftheyieldofindividualampliconsforthevarioustargetscomparedtosingleassayreactions.

4.4 Different amplification ratesDifferentampliconsinRPAcanamplifyatdifferentrates.Undercircumstancesinwhichtargetampliconsmustbeco-amplifiedandtheirexpectedtargetnumbersinasamplearesimilar,thenequalamplificationratesofthetwotargetsmaybeoptimal.However,inothercircumstancesitmaybethecasethatonetargetwillalwaysbeinsignificantexcessovertheother,andsensitivitytothelessabundanttargetmustbemaintained.RPA,incontrasttoPCR,offersadvantagesinthiscaseasitispossibletotuneone’sprimerselectionsanddevelopamultiplexsysteminwhichtheamplificationrateofthelessabundanttargetisfasterthanthemoreabundantspecies.Thisflexibilityisveryusefulwhendetectionofarareeventisdesiredinabackgroundofanabundanttargetwhichistobeusedasaninternalcontrol.

Sample input compatibilityRPAcompatibletemplatehasbeenpreparedfromawiderangeofsources,includingblood,nasalswabs,plantmaterialandculturemedia,bybasiclysismethods(suchasweakalkaliorheattreatment).Oftenfurtherisolationandpurificationofthenucleicacidmaterialpostlysisisunnecessary.RPAisrelativelyrobusttosampleinhibitorsincomparisontoPCR.Thebestapproachhastobespecificallydefinedforeachtypeofapplication(andassay)andisdependentonfactorssuchaspathogentitre,presenceofinhibitorsandlysisrequirements.

5.1 Example lysis methodsSimplemethodssuchasusingnon-ionicdetergents(1%Triton-X100forexample),orheatingcanbeemployed.Sampledilutionwith0.2MNaOHorKOHcanalsoworkwellforlysis.

5.2 Buffer compatibilityBufferscommonlyusedwithPCRwithoutdilution,arealsolikelytobe

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compatiblewithRPA.AlltheobvioussuspectsthatdisruptPCRarealsolikelytoaffectRPA–i.e.EDTA/EGTA,strongdetergents,highsalt,acid/baseetc.

5.3 Termination of amplificationRPAreactionswillrunoutoffuelandreagentseventuallyduringtheamplificationprocess;howeverterminationofamplificationmaybeadesiredfunctionforsomeapplications.Inthosescenariosanumberofstrategiescanbeappliedasfollows:

•heatthereactions(aboveorequalto85oC)todenaturetheproteins.•addEDTAorsimilartochelatetheMgOAcpresent.•dilutethereactionswithwatersothatthecrowdingagentisineffective(atleast1/4dilution).ThismethodisnotrecommendedforTwistAmp®exoorfpgproductsduetothenucleasespresentwhichdegradeampliconovertime.

Other RPA methodsAlthoughnotdirectlysupportedthroughtheinstructionmanuals,manyRPAusershaveappliedthetechniqueinotherformatsfortheirspecificapplications;thoseincludedigital,nesting,microfluidics,solidphase,templategeneration,electrochemistry,colorimetricorSNPdetection.Moreinformationastohowthesemethodshavebeenappliedwith,orincombinationwithRPAcanbefoundinthepublicationslistedattwistdx.co.uk.

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