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1、精品资料Primer-walking(引物步移)........................................精品资料Ifmyinsertislongerthanyourreadlength,howcanIsequenceit?BasicPrimerWalkingOursequencerswillshowyou800-1000ntofunknownsequence,startingfromaknownprimersite.Ifyourunknownregionextendsbeyondthis,youh
2、aveacoupleofoptions.Thediscussionhereisintendedforsituationswheretheunknownsequenceisjustafewkblong.Ifyourunknownislonger-say,10kbormore-thenpleasegotothepageon.****Notethatwedonotperformprimer-walkingorshotgunsequencing.****Primerwalkingistheconsecutivedesignofn
3、ewprimerstoextendintounknownsequence.Ittakes~4daysforeachprimer"step",assumingyousubmitthesamplestotheUMSequencingCoreforsequencing(~2days)andyouorderprimersfromtypicaloligovendors(~2days).Thelongeryouneedto"walk",obviouslythelongerthisprocesswilltake.Forjustafew
4、kb,thisisthebestmethod.Longersequencesmayrequirerandom-fragmentapproaches("shotgun"sequencing).·YouMUSTknowatleastoneregionofsequencefromwhichtostart.Youusethatregion(typicallyavectorsequence)togetthefirstincrementofunknownsequence:·Nowyouhavemore'known'sequence,
5、youcanusethattodesignanewprimerto'walk'furtherintotheunknownregion.Don'tusequestionableregionsofsequencefordesigningthenextprimer!!Seeourwebpageon.........................................精品资料·Continuethatpattern,sequencingwitheachprimer,thendesigningthenext,until
6、youhavesteppedthroughtheentireunknownregion:·Withthatfirst-passdeterminationofyourunknownsequence,youshouldnowsequencetheoppositestrand,inordertoconfirmthesequenceandcorrectanyresidualerrors.Notethattheopposite-strandprimersareplacedinastaggeredfashion,inordertom
7、aximizeyourabilitytogetgoodsequenceatanygivennucleotide:·Afewcommentsaboutthatfinalstep:oSinceyouknowtheentiresequencenow,youcandesignanduse........................................精品资料alltheremainingprimersinonesequencingbatch,forbestspeedandefficiency.oDon'tcoun
8、tongettingidealreadlengthsthroughoutthisprocess.IfyourDNAturnsouttobecontaminated,you'llbesafertohavedesignedtheprimersonly500ntapart.Ifyouwanttogamble,extendt
