Dynamic chemistry for enzyme-free allele discrimination in genotyping by MALDI-TOF mass spectrometry

[摘要] Singlenucleotidepolymorphisms(SNPs)andinsertions/deletions(indels)constituteimportantsourcesofgeneticvariationwhichprovideinsightintodiseaseoriginsanddifferencesindrugresponses.Theanalysisofsuchgeneticvariationreliesuponthegenerationofallele-specificproducts,typicallybyenzymaticextensionoralternativelybythehybridizationofDNAprobes.MALDI-TOFmassspectrometry(MS)hasbeenwidelyusedasaread-outtool.Wedevelopedadistinctenzyme-free,dynamicchemistry-basedmethodofproducingallele-specificproductsforgenotyping.Inablindtrial,peptidenucleicacid(PNA)probesandaldehyde-modifiednucleobaseswereemployedtogenotypetwelvecysticfibrosispatientsfortwomutations(oneSNPandoneindel)linkedtothisdisease.MALDI-TOFMSreportedtheresultingallele-specificproducts.Enzyme-freedynamicchemistrypermittedthegenotypingoftwelveindividualsfortheΔF508(indel)andG551D(SNP)mutations.Nofalsepositivesornegativeswereobserved,andanalysiscouldbeperformedinbothsingleplexandduplexformats.Dynamicchemistryprovidesadistinctmethodofallelediscriminationwithcertainadvantagesoverthosereportedpreviously.AlthoughPCRamplificationisrequired,theanalysisstageisperformedwithoutenzymes,andthereisnoneedforthestringentoptimizationandcontrolofconditionsassociatedwithhybridization-basedmethodsofallelediscrimination...

[学科分类] 分析化学