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1、Downloadedfromgenome.cshlp.orgonAugust11,2018-PublishedbyColdSpringHarborLaboratoryPressResourceConstructionofaBACContigMapofChromosome16qbyTwo-DimensionalOvergoHybridization1CliffS.Han,RobertD.Sutherland,PhillipB.Jewett,MaryL.Campbell,2LindaJ.Meincke,JudyG.Tesmer,MarkO.Mundt,JohnJ.Fawce
2、tt,Ung-JinKim,LarryL.Deaven,andNormanA.DoggettDOEJointGenomeInstitute,BioscienceDivisionandCenterforHumanGenomeStudies,LosAlamosNationalLaboratory,2LosAlamos,NewMexico87545USA;DivisionofBiology,CaliforniaInstituteofTechnology,Pasadena,California91125USAWehaveusedsequence-basedmarkersfrom
3、anintegratedYACSTS-content/somaticcellhybridbreakpointphysicalmapandradiationhybridmapsofhumanchromosome16toconstructanewsequence-readyBACmapofthelongarmofthischromosome.Theintegratedphysicalmapwasgeneratedpreviouslyinourlaboratoryandcontains1150STSs,providingamarkeronaverageevery78kbont
4、heeuchromaticarmsofchromosome16.Theothertwomapsusedforthiseffortweretheradiationhybridmapsofchromosome16fromWhiteheadInstituteandStanfordUniversity.Tocreatelargesequenceabletargetsofthischromosome,weusedasystematicapproachtoscreenhigh-densityBACfilterswithprobesgeneratedfromoverlappingol
5、igonucleotides(overgos).Wefirstidentifiedallavailablesequencesinthethreemaps.Theseincludesequencesfromgenes,ESTs,STSs,andcosmidendsequences.WethenusedBLASTtoidentify36-bpuniquefragmentsofDNAforovergoprobes.Atotalof906overgoswereselectedfromthelongarmofchromosome16.Hybridizationsoccurredi
6、nthreestages:(1)superpoolhybridizationsagainstthe12×coveragehumanBAClibrary(RPCI-11);(2)two-dimensionalhybridizationsagainstrearrayedpositiveBACsidentifiedinthesuperpoolhybridizations;and(3)pooledtertiaryhybridizationsforthoseovergosthathadambiguouspositivesremainingafterthetwo-dimension
7、alhybridization.Forthesuperpoolhybridizations,upto236overgoshavebeenpooledinasinglehybridizationagainstthe12×BAClibrary.Atotalof5187positiveBACsfromchromosome16qwereidentifiedasaresultoffivesuperpoolhybridizations.Thesepositivecloneswererearrayedonmembranesandhybridizedwi
