TheMethylFlash™GlobalDNAMethylation(5-mC)ELISAEasyKit  isacompletesetofoptimizedbuffersandreagentsto colorimetrically quantify globalDNAmethylationstatus byspecificallymeasuringlevelsof5-methylcytosine(5-mC)inasimplified,"one-step"ELISA-likereaction.AsafourthgenerationtechnologyofEpigentek"spatentedglobalDNAmethylationtechnique, itisafurtherrefinementofthepopular,predecessorMethylFlashkitbyimprovinguponspeed,simplicity,sensitivity,andreproducibility.  

Thiskitisalsospecificallyoptimizedforpairedusewiththe MethylFlashGlobalDNAHydroxymethylation(5-hmC)ELISAEasyKit(Colorimetric) forsimultaneouslyquantifyingbothmethylatedDNAandhydroxymethylatedDNA.

Thiskithasthefollowingadvantagesandfeatures:

• Fast -Reducedstepssothattheentireprocedureonlyneeds2hours
• Robust -Improvedkitcompositionallowstheassaytohaveagreater"signalwindow"withreducedvariationbetweenreplicates
• Convenient -Inherentlylowbackgroundnoise,therebyeliminatingtheneedforDNAdenaturationandplateblockingsteps
• Sensitive -Detectionlimitcanbeaslowas0.05%methylatedDNAfrom100ngofinputDNA
• Specific- Highspecificityto5-mC,withnocross-reactivitytounmethylatedcytosineandnocross-reactivitytohydroxymethylcytosinewithintheindicatedconcentrationrangeofthesampleDNA
• Universal - Positiveandnegativecontrolsareincluded andallowfordetectionofDNAmethylationin anyspeciesfromeithersingle-strandedordouble-strandedinputDNA
• Accurate- Optimizedpositivecontrolsthatcanbefractionalizedinpercentagescale,allowingtheassaytobemoreaccurateandhighlycomparablewithHPLC-MSanalysis
• FlexIBLe -Strip-wellmicroplateformatmakestheassayavailableformanualorhighthroughputanalysis

Background
DNAmethylationoccursbythecovalentadditionofamethylgroupatthe5-carbonofthecytosineringbyDNAmethyltransferases,resultingin5-methylcytosine(5-mC).Insomaticcells,5-mCisfoundalmostexclusivelyinthecontextofpairedsymmetricalmethylationofthedinucleotideCpG,whereasinembryonicstem(ES)cells,asubstantialamountof5-mCisalsoobservedinnon-CpGcontexts.Levelsof5-mCarevariableinanimalgenomes,rangingfromundetectableamountsinsomeinsectstoabout2%oftotalDNAinvertebrates.Thelevelof5-mCinplantsgenerallyaccountsfor0.5-2%andcanbeashighas8%oftotalDNAinsomeotherspecies. TheBIOLOGicalimportanceof5-mCasamajorepigeneticmodificationinphenotypeandgeneexpressionhasbeenrecognizedwidely.Forexample,globaldecreasein5-mCcontent(DNAhypomethylation)islikelycausedbymethyl-deficiencyduetoavarietyofenvironmentalinfluences,andhasbeenproposedasamolecularMarkerinmultiplebiologicalprocessessuchascancer.IthasbeenwelldemonstratedthatthedecreaseinglobalDNAmethylationisoneofthemostimportantcharacteristicsofcancer.Afewnovelmodifiednucleotides,5-hydroxymethylcytosine(5-hmC),5-formylcytosine(5-fC)and5-carboxycytosine(5-caC)havebeendetectedinhumanandmousetissuesaswellasembryonicstemcells.Inmammals,thesemodifiednucleotidescanbegeneratedbyiterativeoxidationof5-methylcytosine,areactionmediatedbytheTETfamilyofenzymes.

Principle&Procedure
ThiskitcontainsallreagentsnecessaryforthequantificationofglobalDNAmethylation. Inthisassay,DNAisboundtostrip-wellsthatarespecificallytreatedtohaveahighDNAaffinity.ThemethylatedfractionofDNAisdetectedusingcaptureanddetectionantibodiesandthenquantifiedcolorimetricallybyreADIngtheabsorbanceinamicroplatespectrophotometer.ThepercentageofmethylatedDNAisproportionaltotheODintensitymeasured.

StartingMaterials
InputDNAshouldberelativelypurewith260/280ratio>1.6andcanbedilutedwithwaterorTEbuffer.TheDNAamountcanrangefrom20ngto200ngperreaction.However,werecommendusing100ngofDNA,whichistheoptimizedinputamountforthebestresults. DNAcanbeisolatedfromanyspeciessuchasmammals,plants,fungi,bacteria,andvirusesinavarietyofformsincluding,butnotlimitedto,culturedcells,freshandfrozentissues,paraffin-embeddedtissues,plasma/serumsamples,andbodyfluidsamples.BothsinglestrandedDNAanddoublestrandedDNAwithasizeof200bpstofulllengthissuitableforuse.

WB(10XWashBuffer)
BS(BindingSolution)
NC(NegativeControlwith0%5-mC,50µg/ml)*
PC(PositiveControlcontains5%5-mC,50µg/ml)*
mcAb(5-mCAntibody,1000X)*
SI(SignalIndicator,1000X)*
ES(EnhancerSolution,1000X)*
DS(DeveloperSolution)
SS(StopSolution)
8-WellAssayStrips(WithFrame)
UserGuide

*Spinthesolutiondowntothebottompriortouse.

Note:TheNC isunmethylatedDNAcontaining0%of5-methylcytosine.ThePCismethylatedDNAcontaining5%of5-methylcytosine.