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1、GENOMICS52,1±8(1998)ARTICLENO.GE985423AnImprovedApproachforConstructionofBacterialArti®cialChromosomeLibraries12KazutoyoOsoegawa,PengYeongWoon,BaohuiZhao,EirikFrengen,34MinakoTateno,JosephJ.Catanese,andPieterJ.deJongDepartmentofGenetics,RoswellParkCancerInstitute,ElmandCarltonStreet
2、s,Buffalo,NewYork14263ReceivedFebruary24,1998;acceptedJune3,1998nomes(Chumakovetal.,1992;Footeetal.,1992).Presentedhereareimprovedmethodologiesthaten-However,theyareoftenunstableandalsoadif®cultablethegenerationofhighlyredundantbacterialar-sourceforobtainingpureDNAinsuf®cientquantit
3、iesti®cialchromosome/P1-derivedarti®cialchromosomeforthepreparationofthesmall-fragmentlibrariesre-libraries,withlargerandrelativelyuniforminsertquiredforDNAsequencing.Inrecentyears,bacterialsizes.Improvementsinvectorpreparationanden-arti®cialchromosome(BAC)(Shizuyaetal.,1992)andhanc
4、edligationconditionsreducethenumberofback-P1-derivedarti®cialchromosome(PAC)(Ioannouetal.,groundnonrecombinantclones.Preelectrophoresisof1994)cloneshaveprovidedabetteralternativeforimmobilizedhigh-molecular-weightDNAremovesin-mammaliangenomemappingandsequencing(Venterhibitorsofthecl
5、oningprocess,whilesizingDNAfrag-etal.,1996;Marraetal.,1997;Gregoryetal.,1997).mentstwicewithinasinglegeleffectivelyeliminatesAlthoughthebacterialcloneshaveinsertsizesconsid-smallrestrictionfragments,thusincreasingtheaver-erablysmallerthanthoseofYACs,theyarerelativelyageinsertsizeoft
6、heclones.Thesize-fractionatedstable,andtherecombinantDNAcanbeisolatedbyDNAfragmentsarerecoveredbyelectroelutionrathersimpleplasmidextractionprocedures.Notsurprisinglythanthemorecommonmeltingofgelsliceswithsub-sequentb-agarasetreatment.Concentrationoftheli-therefore,BACsandPACshavebe
7、comethemainsub-gationproductsyieldsa6-to12-foldreductioninthestratesforhumangenomesequencing.Severalproce-numberofelectroporationsrequiredinpreparingaduralaspectsthatimprovethequalityandef®ciencyoflibraryofdesirablesize.Theseimprovedmethodsthecloningprocesshavebeenaddressed.Asaresul
8、t,havebeenappliedto
