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.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。