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Molecular Analysis and Results DNA
TheoryofCGHComparativegenomichybridization(CGH)isafairlynewmolecularcytogenetictechniquethatallowsdetectionofDNAsequencecopynumberchangesthroughoutthegenomeinasinglehybridization.CGHisbasedonco-hybridizationofdifferentiallylabeledtumorandnormalDNAtohumanmetaphasechromosomes.Inatypicalexperiment:
- ThetumorDNAislabeledwithFITC-dUTPandnormalDNAwithTexasRed-dUTPpriortohybridization.
- Threeimagesofthehybridization,representingbothfluorescentlabelsaswellasDNAcounterstain(DAPI),areacquiredusingepifluorescencemicroscopyandaCCDcamerainterfacedtoacomputer.
- Subsequently,animageanalysissoftwareprogramisusedtocalculategreen-redfluorescenceintensityratioprofilesalongallchromosomes.
- RegionsofthegenomethatareeithergainedorlostinthetumorareindicatedbythedifferencesbetweenbindingofthetwolabeledDNAsasevidencedbythefluorescenceintensityratioprofiles.
Thus,inasinglehybridizationitispossibletoscreenallchromosomalsitesthatmaycontaingenesthatareeitherdeletedoramplifiedincancer.
SensitivityThesensitivityofCGHindetectinggainsandlossesofDNAsequencesisapproximately2-20Mb.Forexample,a10-foldamplificationofa200kbregionshouldbedetectableunderoptimalhybridizationconditions.EarlyCGHexperimentsweredoneusingDNAisolatedfromfreshlyfrozentumortissues.However,ithasnowbeenshownthatDNAextractedfromformalin-fixedparaffin-embeddedarchivaltissueblockscanbeusedaswell.PriortoCGHhybridization,DNAcanbeuniversallyamplifiedusingdegenerateoligonucleotide-primedPCR(DOP-PCR),whichallowstheanalysisof,forexample,microdissectedtumorsamples.Thelattertechniqueis,however,moredifficultandlessreliablethanCGHwithoutPCRpre-amplificationstep.
ApplicationsofCGHCGHhasbeenusedtodetectgeneticaberrationsinavarietyofmalignancies:
- Ithasalreadyrevealedacomplexgeneticnatureofcancerandindicatedseveralnovelchromosomalregionsthatarefrequentlyalteredintumors.
- SeveralCGHstudiesonprostatecancerhavebeenpublished,e.g.,lossof6q,8p,13q,and16q,aswellasgainsof7p,7q,8qandXq,havebeenfoundtobecommonalterationsinprostatecancer.
- CGHfindingsledalsotothediscoveryofandrogenreceptor(AR)geneamplificationinhormone-refractoryrecurrentprostatecarcinomas.
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