Protocol
FortheoriginalprotocoldescribingCGHseethearticlebyKallioniemietal.ForanexampleofhowthistechniquehasbeenutilizedbytheNCIProstateCancerWorkingGroupseethesectiononCellLines.
DNARNAProteomic
DNAsequencecopynumberchangesthroughoutthegenomeinasinglehybridization.CGHisbasedonco-hybridizationofdifferentiallylabeledtumorandnormalDNAtohumanmetaphasechromosomes.
Inatypicalexperiment:
ThetumorDNAislabeledwithFITC-dUTPandnormalDNAwithTexasRed-dUTPpriortohybridization.
Threeimagesofthehybridization,representingbothfluorescentlabelsaswellasDNAcounterstain(DAPI),areacquiredusingepifluorescencemicroscopyandaCCDcamerainterfacedtoacomputer.
Subsequently,animageanalysissoftwareprogramisusedtocalculategreen-redfluorescenceintensityratioprofilesalongallchromosomes.
RegionsofthegenomethatareeithergainedorlostinthetumorareindicatedbythedifferencesbetweenbindingofthetwolabeledDNAsasevidencedbythefluorescenceintensityratioprofiles.
Thus,inasinglehybridizationitispossibletoscreenallchromosomalsitesthatmaycontaingenesthatareeitherdeletedoramplifiedincancer.
DNAsequencesisapproximately2-20Mb.Forexample,a10-foldamplificationofa200kbregionshouldbedetectableunderoptimalhybridizationconditions.EarlyCGHexperimentsweredoneusingDNAisolatedfromfreshlyfrozentumortissues.However,ithasnowbeenshownthatDNAextractedfromformalin-fixedparaffin-embeddedarchivaltissueblockscanbeusedaswell.PriortoCGHhybridization,DNAcanbeuniversallyamplifiedusingdegenerateoligonucleotide-primedPCR(DOP-PCR),whichallowstheanalysisof,forexample,microdissectedtumorsamples.Thelattertechniqueis,however,moredifficultandlessreliablethanCGHwithoutPCRpre-amplificationstep.