Applications

• Preparationofcompleteandunbiasedfosmidlibraries,fromanyBIOLOGicalsample,thatcanbemaintainedatsingle-copynumberandinducedtohigh-copynumberasneeded,usingtheCopyControl™AutoinductionSolution.
• Constructionofmetagenomiclibrariesfrommicrobespresentinenvironmentalsamplessuchaswaterandsoil.

TheCopyControl™FosmidLibraryProductionKits*provideanefficientandimprovedmethodforconstructingalibraryofapproximately40kbclones.TheCopyControlpCC1FOS™VectorcontainsboththeE.coliF-factorsingle-copyoriginofreplicationandtheinducIBLehigh-copyoriV(Fig.1).CopyControlFosmidclonesaretypicallygrownatsinglecopytoensureinsertstabilityandsuccessfulcloningofencodedandexpressedtoxicproteinandunstableDNAsequences.TheCopyControlFosmidclonescanthenbeinducedupto50copiespercellimmediatelybeforeDNApurification.ThisstepgreatlyincreasesDNAyields,whilemaintainingthestabilityoftheplasmid.

TheCopyControlHTPFosmidLibrarycontainsthepCC2FOS™Vectorwhichisdesignedtooptimizeend-sequencingresults,especiallyinahigh-throughputsetting.Theprimercassette,engineeredinconjunctionwithAgencourtBioscienceCorporation,eliminateswastefulandexpensivevectorsequencereadsbyhavingthe3´endsoftheprimer-annealingsitesonlythreebasesfromthevector/insertjunction.Inaddition,theseven-basesequenceatthe3´endofeachprimerwasspecificallydesignedtominimizemisprimingtoanycontaminatingE.coliDNApresentaftertemplatepurification(Fig.2).

Thekitusesastrategyofcloningblunt-endedDNAfragmentsgeneratedbyrandomshearingoftheDNA,toproducemorecompleteandunbiasedgenomiclibrariesthancanbeobtainedbypartialrestrictionendonucleasedigests.GenomicDNAisfirstshearedintoapproximately40-kbfragments.TheshearedDNAisend-repairedtogenerateblunt,5´-phosphorylatedendsandthensize-selectedbyandrecoveredfromalow-melting-pointagarosegel.Finally,thesize-selectedDNAisligatedintothecloning-readyCopyControlpCC1FOSorpCC2FOSVector,packagedusingultra-highefficiencyMaxPlax™LambdaPackagingExtracts(>10⁹pfu/µgDNA),includedinthekit,andplatedonthesuppliedTransforMax™EPI300™E.coli(Fig.3).

Figure1.CopyControl™Vectormap.TheCopyControlpCC1FOS™andpCC2FOS™VectorsforCopyControlFosmidlibraryproductionaresuppliedlinearizedattheEco72I(blunt)siteandthendephosphorylated.Thevectorisreadyforcloningend-repaired(blunt-end)genomicDNAofapproximately40kb.

Figure2.TheCopyControl™pCC2FOS™Vectorprimercassette.ThevectordiffersfromthepCC1FOSVectorbytheengineeringofanewprimercassettethateliminateswastefulvector-derivedsequencingreadsandminimizesthepotentialforprimingontheE.coligenome.

Benefits

• CopyControlpCC1FOSandpCC2FOSVectorsaresuppliedCloning-Ready:linearized,dephosphorylated,purified,andreadyforligation.
• NoneedforpartialrestrictionendonucleasedigestsorpulsefieldgelelectrophoresistopreparethegenomicDNAforcloning.
• Maximizehigh-throughputend-sequenceresultsusingthepCC2FOSVector(Fig.4).
• Clonescanbeinducedfromsinglecopyupto50copiespercell(Fig.5).SafelyobtainhigherDNAyieldswhilemaintainingthestabilityofsingle-copy-numberclones.
• High-efficiencylambdapackagingeliminatesbackgroundandfalsepositives.
• "Hands-off"autoinductionprotocolisperfectforhigh-throughputapplications.
• FasterandeasierthanBACcloning.

Figure3(clicktoenlarge).OverviewoftheprocessforpreparingafosmidlibraryusingtheCopyControl™FosmidLibraryProductionKits.Oncethelibraryhasbeenprepared,individualclonescanbeculturedinsmallvolumeandinducedtomultiple-copynumberforhighyieldsofhigh-purityDNAforfingerprinting,sequencing,etc.,usingEpicentre'sDirectLysisFosmid96kitorFosmidMAX™DNAPurificationKit.

Figure4.TypicalsequencingresultsobtainedwiththepCC2FOS™ForwardPrimeronapCC2FOS™cloneat1/48xBigDye™dilution.SimilarresultswereobtainedwiththepCC2FOSReversePrimer(datanotshown).

Citations

1. FEMSMicrobiolLett284(2008)28-34

*Coveredbyissuedand/orpendingpatents.