TheMethylFlash™HydroxymethylatedDNAQuantificationKit(Fluorometric)isacompletesetofoptimizedbuffersandreagentstofluorometricallyquantifyglobalDNAhydroxymethylationbyspecificallymeasuringlevelsof5-hydroxymethylcytosine(5-hmC)withoutcross-reactivitytomethylcytosineandunmethylatedcytosine. Thiskitisalsospecificallyoptimizedforpairedusewiththe MethylFlashMethylatedDNAQuantificationKit(Fluorometric) forsimultaneouslyquantifyingbothmethylatedDNAandhydroxymethylatedDNA. About5-Hydroxymethylcytosine
5-hmCisamodifiedformofcytosine,recentlydiscoveredinanimaltissues.Thefunctionof5-hmCinepigeneticsmaybedifferentfromitsforerunner5-methylcytosine(5-mC)andcurrentlyremainsamystery.Itisbelieved,though,that5-hmCplaysanimportantroleinswitchinggenesonandoff. Thepresenceof5-hmCmakesitnecessarytonotonlyre-evaluateexistingDNAmethylationdata,butalsonecessarytodeterminetherelativedistributionandchangesof5-hmCinhumantissuesofhealthyanddiseasedstatuses.PriortoEpigentek’sMethylFlash™ technology,therewerenomethodsthatcouldbeusedforpracticallyandroutinelyidentifying5-hmCanddiscriminatingthisbasefrom5-mC. DistinguishingBetween5-hmCand5-mC
CurrentlyusedmethylatedDNAanalysismethodsincludingrestrictionenzymedigestionandbisulfiteorMeDIP-mediatedMS-PCRandsequencingarenotsuitablefor5-hmCdetectionas5-hmCand5-mCarevirtuallyindistinguishablebetweeneachotherwiththesemethods.Toaddressthisproblem,EpigentekofferstheMethylFlash™HydroxymethylatedDNAQuantificationKit(Fluorometric)whichusesauniqueproceduretoquantifyglobalDNAhydroxymethylation. Thisproductprovidesacost-effectivewaytomeasurelevelsof5-hydroxymethylcytosineandtodistinguishbetween5hmC,5mC,andC.Thisallowsforresearcherstore-evaluatetheirDNAmethylationdataforDNAhydroxymethylationandtoefficientlylookforDNAhydroxymethylationinnewDNAsamples. Thekithasthefollowingadvantagesandfeatures: - Fluorometricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbecompletedwithin3hoursand20minutes.
- Highsensitivity,ofwhichthedetectionlimitcanbeaslowas10pgofhydroxymethylatedDNAand acceptsaslowas20ngofinputgenomicDNA
- Highspecificitywithnocross-reactivityto methylcytosineand unmethylatedcytosine.5-hydroxymethylcytosineisseparatelydetected.
- Universalpositiveandnegativecontrolsareincluded,whicharesuitableforquantifyinghydroxymethylatedDNAfromanyspecies.
- Strip-wellmicroplateformatmakestheassayflexIBLeforeithermanualorhighthroughputanalysis.
- Simple,reliable,andconsistentassayconditions.
ComparisonofAvailableELISA-basedGlobalDNAMethylationAssayMethods | | MethylFlash (Fluorometric) | CompetitorZ |
|---|
| AssayPrinciple | DirectELISA-Detectionantibodyspecificallyanddirectlydetects5-hmC | IndirectELISA-DetectionantibodydetectsdsDNA,(detectedsignalsisdependentonlyonintactDNAstrandsanddoesnotquantifyactualtotal5-hmC) | | Format | 96-wellplate | 96-wellplate | | Sensitivity | Excellentdetectionlimitof0.01ngof5-hmCDNA | Unclear,asthedetectedsignalsarefromintactDNAstrands | | S/NRatio | >20withverylowbackground | <4withhighbackground | | ProceduralConvenience | NoneedforplateblockinganddenaturationofDNA | RequiresplateblockingandDNAdenaturation | | ProtocolTime | <4hr | <5hr | | DNAType&Species | Universalforanyspecies,bothssDNAanddsDNA | OnlyssDNAfromlimitednumberofspecies | | Specificity | High,theantibodyonlydetects5-mC | Unclear,astheassayprincipleisnotreasonable | | QuantificationType | Bothrelativeandabsolutequantification | Relativeonly | | StandardControl | Stable,quantitativeinabsoluteamountof5-mC,andcanbeuniversallyusedforanyspecies | Unclear | | MinimumInputAmount | 20ng | 100ng | | AccuracyofDetection | StableandquantifiedstandardcontrolwithprovenclosecorrelationwithHPLC-MSanalysisbyusers | Unclear,basedonbiasedassayprinciple | | %5-mCCalculation | Simple | Complicated | | PatentedMethod | Yes | No | | Popularity | Veryhighpublishedcitationcount | Lowpublishedcitationcount | | SupportExpertise | Since2006 | Since2012 |
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 Fig.1.SchematicprocedureoftheMethylFlash™HydroxymethylatedDNAQuantificationKit. |
