Description

SingleprimerPCRallowsamplificationfromknowntounknownregionsinchromosomes,phage,plasmids,largePCRproductsandothersourcesofDNA.

Atsufficientlylowstringency,anyprimerwillmisprimewhilecontinuingtobindspecificallytoitsintendedsite.Conditionscanusuallybefoundallowingmisprimingsufficientlyclose(<3.5kb)tothecorrectsitetopermitamplificationanchoredatthesame.

Reamplificationwithanestedprimerandtheoriginaloutsideprimergeneratesaproductwithuniqueends.Theresultingsizeshiftcanbeusedtodiagnosethecorrectproduct,whichcanthenbesequencedfromeitherend.

Twomethodsarepresented.The"Short"methodcanbedonequicklyandworksabout80%ofthetimeinourlabforanygivenprimerPo(seefigurebelow)IthasbeenadaptedfromHermannetal(1).The"Long"method,developedinourlaboratory(2),requiresmoretimebutwilloftenworkwhentheshortmethodfails.Anotheradvantageofthelongmethodisthattheproducthasuniqueends,allowingconvergentsequencing.

WhenamplifyingoutofinvertedrepeatsattheendsofcertainelementssuchasTn10orTn5,gelpurifybandsbeforesequencing.IfyoutrytosequencethecrudePCRreaction,youwillgetsuperimposedsequencescomingoutofbothends.

Inourlab,PCRisalwaysdoneinanAirThermoCycler(IdahoTechnologies,POBox50819,IdahoFalls,ID83402),whichrampsquickly,allowingshortdwelltimesduringdenaturationandannealing.Thisenablesrapidandstringentpolymerization,whileminimizingenzymeagingandtemplatehydrolysis.Wehavenottestedtheprotocalonothermachines.

Inanappendixattheend,Ipresentsomeprimerswhichhaveworkedwellinourlabwhensequencingoutofcommoninsertionsequences.

Protocol

SingleprimerPCRworksbestwhenwhentherearenestedprimersPoandPnwithsitesintheknownregion:

1.ShortMethod

Whenthismethodfails,tryvaryingtheannealingtemperatureinthesecondcyclingregime.Ifthatdoesn"twork,trychangingprimers.Ifproblemspersist,switchtothelongmethod.

DothefollowingPCRreaction:

15µLH2O3µL10xM-PCRB3µL4dNTPs@2mMeach3µLPo@5µM3µLtemplatedilutedtoabout1ng/µL3µL"T/TS"@0.4uTaq/µL--------30µL

Cycleasfollows:

30"x94°C20cycles0"x94°C0"x55°C1"x72°S=930cycles0"x94°C0"x40°C1"x72°S=630cycles0"x94°C0"x55°C1"x72°S=9

Cleanup:

Add1µLExoInuclease@1u/µL1hrx37°PurifyusingQiaquick(orrelated)technology,elutein30µLTECheck3µLon1%agarosegelSequencewithprimerPn

2.LongMethod

StringencyoptimizationisdonewithPo,andreamplificationusingabiasedratioofPoandPn.

Make3reactionswithvarying[Mg++]:

15µLH2O3µL10xH,MorL-PCRB3µL4dNTPs@2mMeach3µLPo@5µM3µLtemplatedilutedtoabout1ng/µL3µL"T/TS"@0.4uTaq/µL--------30µL

Distribute310µLaliquotsofeachmixintocapillariesanddiscardremainder.Loadsetsof3capillaries,onefromeachmix,consecutivelyintothesamemachinewiththeannealingtemperature("Ta")setat40,45and50°,respectively.

Cycleasfollows:

30"x94°C20cycles0"x94°C0"x55°C1"x72°S=930cycles0"x94°C0"xTa1"x72°S=630cycles0"x94°C0"x55°C1"x72°S=9

Loada0.8%agarosegelaccordingtothefollowingpattern:

Reactionsarrayedinthisfashionhaveroughlyincreasingstringencyfromlefttoright.

FindthehigheststringencyatwhichdistinctbandsarevisIBLe.Usually,allsuchbandsareanchoredatthespecificsitecorrespondingtoPo.Thisisespeciallytrueifthereareonlyoneortwo,thesituationwewish.

IsolateDNA.Wegenerallycorethebandwithayellowtip,andsoakitovernightat4°inasmallamountofTEorH2.Thesupernatantprovidesthetemplateforreamplification.

Itisalsousefultoreamplifytheunfractionatedproductsofthefirstamplification,afterfirstremovingprimersbyQiaquick,Wizard,orsimilarmethodology.Runningthefirstandsecondamplificationproductssidebysidewillrevealcorrectlyanchoredbands,astheywillbeshiftedwithrespecttoparentalbands.

Reamplification

40µLH2O10µL10xM-PCRB10µL4dNTPs@2mMeach10µLPnprimer@5µM10µLPoprimer@0.2µM10µLDNAfrompart110µL"T/TS"@0.4uTaq/µL---------100µL

Loadglasscapillariesandamplify:

30"x94°C30-40cycles0"x94°C0"x55°30"x72°5"x72°

IhaveassumedtypicalvaluesforTaandextensiontime.ThesemaybemodifiedasrequiredbyPnandproductsize.IgenerallyletTaequal10°lessthantheprimerTm.15-30"issufficientforproductsupto1kbintheAirCycler.

Electrophoresea5µLaliquotasbefore.Becausethereamplificationwasdonewitha25:1ratioofPntoPo,thesmallerbandshoulddominate,andmaybetheonlyonevisible.Ifthecontaminantbandisnotpresentorhasayieldonlyasmallfractionofthatofthesmallerband,thentheremaining95µLofproductcanbecleanedupdirectlyusingaWizardPCRpurification.Otherwise,runtheentirereactiononanagarosegelandpurifyDNAfromtheappropriateband.

Theprepisnowreadytobesequenced.Pnwillprimefromtheknownend,andPofromtheunknownend.

Materials

1.10xH,MorL-PCRB

500mMTris,pH8.32.5mg/mLBSAMgCl2togive30,20or10mM,respectively5%Ficoll5mMcresolred

2.T/TS

10.5µLEDB1µLTaqPolymerase(Promega)@5u/µL1µLTaqStartantibody(CloneTech)asdelivered

3.EDB

2.5mg/mlbovineserumalbuminin10mMTris,pH8.3

References

1.S.R.J.A.M.Hermann,S.O"Neill,T.T.Tsao,R.M.Harding&J.L.Dale"Singleprimeramplificationofflankingsequences"Biotechniques29,1176-1180(2000)

2.E.C.Kofoid,C.Rappleye,I.Stojiljkovic&J.Roth,"The17-geneethanolamine(eut)operonofSalmonellatyphimuriumencodesfivehomologuesofcarboxysomeshellproteins"J.Bacteriol.181,5317-5329(1999)

Appendix--SomeUsefulPrimers

Primercommonnamesaregivenfirstandshouldnotbeoverinterpreted.Databasenamesareinparentheses,followedbyacomment.Sequencesarewritten5"to3".Eachprimerextendsoutofitsassociatedcassettebytheshortestpossibleroute(thatis,itemanatesoutof--notinto--thecassette).

1.Tn10dTetcoreandderivatives

Po:TN10L(TP633)BindsjustaftertetAterminator.ACCAACCATTTGTTAAATCAGTTTTTGTTGTGAPo:TN10R(TP632)BindsjustaftertetRterminator.CAGTGATCCATTGCTGTTGACAAAGGGAATCPn:AnyappropriateIS10primerbelow.

2.IS10andmostTN10derivatives

Po:F1284(TP89)38bpfromend;willnotworkinTPOPs;CAAGATGTGTATCTACCTTAACPoorPn:IS10R2(TP134)27bpfromend;rightsideofTPOPsonly;CAAGATGTGTATCCACCTTAACTTAATGATTTTPn:IS10R4(TP134)4bpfromend;anyTn10derivative,includingTPOPs;AACTTAATGATTTTGATCAAAATCATTAGGGGATTCA

3.MudJandrelatives

Po:R86(TP251)61bpbeforeleftend.GCAAGCCCCACCAAATCTAATCCCAPn:R54(TP240)36bpbeforeleftend.CCGAATAATCCAATGTCCPo:F33(TP81)9bpfromend;extremelydifficultPCR,becauseoflargestem-loop;thefirst5cyclesshouldeachbeprecededbya5"holdat94°Cinsteadofthenormalzerosecondholds;statistically,halfthebandswillbeproductsextendingintothemudelement,notout.GAAACGCTTTCGCGTTTTTCGTGCGPn:F20(TP79)Exactlyatend.GTTTTTCGTGCGCCGCTTC

4.Tn5-derivedchloramphenicolresistancecassette(foundinMud-camandTn10d-cam)

Po:CMR2(TP699)158bppriortogenefacingupstream.CTTCCCGGTATCAACAGGGACAPn:CMR1(TP698)214bppriortogenefacingupstream.GTCACAGGTATTTATTCGGCGCAPo:CKO3(TP45)154bppastgenefacingdownstream.AGGGCAGGGTCGTTAAATAGCPn:CMR3(TP700)223bppastgenefacingdownstream.AGTGTGACCGTGTGCTTCTCAA