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1、Downloadedfromgenome.cshlp.orgonAugust13,2018-PublishedbyColdSpringHarborLaboratoryPressPCR-basedApproachesforDetectionofMutatedrasGenesAlphonsusD.M.vanMansfeldl,2andJohannesL.BosI1LaboratoryforPhysiologicalChemistryand2InstituteofMolecularBiologyandMedicalBiotechnology,UniversityofUtrecht,352

2、1GGUtrecht,TheNetherlandsTherasgenesencode21-kDGTP-Allele-specificOligonucleotidedifferent,labeledoligonucleotides,bindingproteins(p21ras)thatcyclebe-HybridizationeachofwhichcorrespondstooneoftweenaninactiveGDP-boundformAllele-specificoligonucleotidehybrid-theexpectedpointmutationsintheandanac

3、tiveGTP-boundform.Theization(ASO)todetectpointmuta-genesegment.Hybridizationofoneofp21rasproteinsarethoughttoplayantionsisbasedonthefactthatanoligo-theseallele-specific(pointmutation-importantroleinsignaltransductionnucleotidewithonemismatchbindsspecific)oligonucleotidesshowsthethateventuallyc

4、ontrolsproliferationmoreweaklytoits(imperfect)comple-presenceofthecorrespondingpointanddifferentiationofmanydifferentmentthanaperfectlymatchingone.mutation.Hybridizationandstringentcells.Theras(proto-onco)genescanbeByperforminghybridizationandwash-washingmaybeperformedinthepres-activatedasonco

5、genesbysimplepointingjustbelowthedissociationtemper-enceof3Mtetramethylammoniummutations,whichsubstituteasingleature(Tm)oftheperfectlymatchingchloride.O)Atthisconcentrationtheaminoacidresidueatacriticalpositionoligonucleotide,onecandiscriminateG-CandA-Tbasepairshavesimilarintheproteinproduct.M

6、utatedrasbetweenaperfectlymatchingoligonu-stabilities,sothatoligonucleotidesofgenesoccurwithahighfrequencyincleotideandanoligonucleotidewith1-thesamelengthhavethesameTm,re-commonhumancancers,includingbpmismatch.(2)Inshort,PCR-ampli-gardlessofthebasecomposition.Thisadenocarcinomaofthelung,color

7、ectalfledDNAisspottedontoanylon(orallowshybridizationandwashingofcancer,myeloidleukemia,andadeno-nitrocellulose)filterandhybridizedtothefilterswithdifferentoligonucleo-carcinomaofthepancreas.O)aradioactivelylabeledoligonucleotidetidesne