Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences 英文参考文献.docVIP

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Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences 英文参考文献.doc

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Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences 英文参考文献

FastandNon-ToxicInSituHybridizationwithout BlockingofRepetitiveSequences SteenH.Matthiesen1*,CharlesM.Hansen2 1ResearchandDevelopment,Dako,Glostrup,Denmark,2CharlesM.HansenConsulting,H?rsholm,Denmark Abstract Formamideisthepreferredsolventtolowerthemeltingpointandannealingtemperatureofnucleicacidstrandsininsitu hybridization (ISH). A key benefit of formamide is better preservation of morphology due to a lower incubation temperature. However, in fluorescence in situ hybridization (FISH), against unique DNA targets in tissue sections, an overnight hybridization is required to obtain sufficient signal intensity. Here, we identified alternative solvents and developed a new hybridization buffer that reduces the required hybridization time to one hour (IQFISH method). Remarkably,denaturationandblockingagainstrepetitiveDNAsequencestopreventnon-specificbindingisnotrequired. Furthermore,thenewhybridizationbufferislesshazardousthanformamidecontainingbuffers.Theresultsdemonstrate asignificantincreasedhybridizationrateatalowereddenaturationandhybridizationtemperatureforbothDNAandPNA (peptide nucleic acid) probes. We anticipate that these formamide substituting solvents will become the foundation for changesintheunderstandingandperformanceofdenaturationandhybridizationofnucleicacids.Forexample,theprocess time for tissue-based ISH for gene aberration tests in cancer diagnostics can be reduced from days to a few hours. Furthermore, the understanding of the interactions and duplex formation of nucleic acid strands may benefit from the propertiesofthesesolvents. Citation: Matthiesen SH, Hansen CM (2012) Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences. PLoS ONE 7(7): e40675. doi:10.1371/journal.pone.0040675 Editor:KonradinMetze,UniversityofCampinas,Brazil ReceivedMarch6,2012;AcceptedJune12,2012;PublishedJuly24,2012 Copyright:?2012Matthiesen,Hansen.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits