Primer-walking(引物步移)

1Ifmyinsertislongerthanyourreadlength,howcanIsequenceit?BasicPrimerWalkingOursequencerswillshowyou800-1000ntofunknownsequence,startingfromaknownprimersite.Ifyourunknownregionextendsbeyondthis,youhaveacoupleofoptions.Thediscussionhereisintendedforsituationswheretheunknownsequenceisjustafewkblong.Ifyourunknownislonger-say,10kbormore-thenpleasegotothepageonshotgunsequencing.****Notethatwedonotperformprimer-walkingorshotgunsequencing.****Primerwalkingistheconsecutivedesignofnewprimerstoextendintounknownsequence.Ittakes~4daysforeachprimerstep,assumingyousubmitthesamplestotheUMSequencingCoreforsequencing(~2days)andyouorderprimersfromtypicaloligovendors(~2days).Thelongeryouneedtowalk,obviouslythelongerthisprocesswilltake.Forjustafewkb,thisisthebestmethod.Longersequencesmayrequirerandom-fragmentapproaches(shotgunsequencing).YouMUSTknowatleastoneregionofsequencefromwhichtostart.Youusethatregion(typicallyavectorsequence)togetthefirstincrementofunknownsequence:2Nowyouhavemore'known'sequence,youcanusethattodesignanewprimerto'walk'furtherintotheunknownregion.Don'tusequestionableregionsofsequencefordesigningthenextprimer!!SeeourwebpageonDesignofSequencingPrimers.Continuethatpattern,sequencingwitheachprimer,thendesigningthenext,untilyouhavesteppedthroughtheentireunknownregion:3Withthatfirst-passdeterminationofyourunknownsequence,youshouldnowsequencetheoppositestrand,inordertoconfirmthesequenceandcorrectanyresidualerrors.Notethattheopposite-strandprimersareplacedinastaggeredfashion,inordertomaximizeyourabilitytogetgoodsequenceatanygivennucleotide:Afewcommentsaboutthatfinalstep:oSinceyouknowtheentiresequencenow,youcandesignandusealltheremainingprimersinonesequencingbatch,forbestspeedandefficiency.oDon'tcountongettingidealreadlengthsthroughoutthisprocess.IfyourDNAturnsouttobecontaminated,you'llbesafertohavedesignedtheprimersonly500ntapart.Ifyouwanttogamble,extendthespacingto700nt.oPleaserefertotheDesignofPrimersforAutomatedSequencingguideforinformationabouthowtodesignsuccesfulsequencingprimers.4oYouMAYknowsomevectorsequenceforstartingtheoppositestrand(startingattherightendoftheinsert,progressingtowardstheleft).Ifso,considerwalkingfromthatsideatthesametimeasyouwalkfromtheleft,andyou'llcutthewalkingtimeinhalf.