TheEpiQuik™HydroxymethylatedDNAImmunoprecipitation(hMeDIP)Kit isacompletesetofoptimizedbuffersandreagentstocarryoutasuccessfulhMeDIPprocedurefortheselectiveenrichmentofDNAfragmentscontaining5-hydroxymethylcytosine(5-hmC)inahighthroughputformatusingDNAisolatedfromanyspecies.ThehydroxymethylatedDNAthatisenrichedwiththiskitcanbeusedinvariousdownstreamapplicationsincludingPCR(hMeDIP-PCR)andmicroarrays(hMeDIP-chip)forinvestigatinggene-specifichydroxymethylationincellsortissues.

About5-Hydroxymethylcytosine
5-hmCisamodifiedformofcytosine,recentlydiscoveredinanimaltissues,and playsanimportantanddifferentrolethan5-mCinregulationofDNAmethylation,chromatinremodeling,andgeneexpression,particularlyinbrain-specificgeneregulation.Quiterecently,5-hmCwasobservedtobelinkedtoepigeneticreprogramminginmammalianzygotes.However,theexactfunctionsof5-hmChavenotyetbeenfullyidentifiedsincegene-specificdistributionof5-hmCisunknownduetotheinABIlityofcurrentlyusedDNAmethylationanalysismethodstodistinguishbetween5-hmCand5-mC.

ProductFeatures
Becauseofthepresenceof5-hmCinDNAwithunclearfunctionsingeneregulationandbecauseofthediscoveryofenzymesthatproduce5-hmC,itiscrucialtoidentifyhydroxymethylationstatusinspecificgeneloci,whichwouldhelptobetterunderstandmethylation-basedepigeneticregulationofgenefunctions.Toachievethis,EpigentekhasdevelopedaninnovativemethodtocaptureDNAfragmentscontaining5-hmC,andincorporatedthismethodintoitsEpiQuik™HydroxymethylatedDNAImmunoprecipitation(hMeDIP)Kit.Thiskitusesahighaffinity5-hmCantibodytoselectivelycapturedouble-strandedorsinglestrandedDNAfragmentscontaining5-hmC.Thekithasthefollowingfeatures:

• Extremelyfastandconvenientprotocol withatotalproceduretime(frominputsampletoready-to-usehydroxymethylatedDNA) oflessthan3hours,whichincludes aminimalhandlingtimeoflessthan20minutes.
• FlexIBLe96stripwellmicroplateformatmakestheassayveryeasytohandle:manualmethodwithonereactionatatimeorhighthroughputmethodwith96reactionsatatime.
• Highlyefficientenrichmentratioofpositive/negativecontrol>1000.
• LowDNAinputrequirementofaslowas0.1µgperreaction.
• Highreproducibilitythroughpre-optimizedhMeDIPconditions.
• CompatiblewithvariousdownstreamanalysisworkflowsincludinghMeDIP-PCRandhMeDIP-chip.

PrincipleandProcedure
TheEpiQuik™HydroxymethylatedDNAImmunoprecipitation(hMeDIP)KitcontainsallreagentsrequiredforcarryingoutasuccessfulhMeDIPprocedureusingDNAisolatedfrommammaliancellsortissues.ThiskitincludesapositivecontrolDNAfragment,anegativecontrolnon-immuneIgG,andcontrolprimersthatcanbeusedwiththepositivecontroltodemonstratetheenrichmentefficacyforhydroxymethylatedDNAwiththekitreagentsandprotocol.ThepositivecontrolDNAcontaining5-hmCcanbeimmunoprecipitatedbya5-hmCantibodybutnotbyanon-immuneIgG.InthishMeDIP,immunoprecipitationof5-hmC-enrichedDNAfragmentsisprocessedinamicroplateunderoptimizedreactionconditions,whichenableshMeDIPtobecompletedwithin3hourswithhighefficiency.ImmunoprecipitatedhydroxymethylatedDNAisthencleaned,released,andeluted.ElutedDNAcanbeusedforvariousdownstreamapplicationsincludingPCR(hMeDIP-PCR)andmicroarray(hMeDIP-chip).

Easy,Fast,andFlexible
TheentirehMeDIPprocedurehaseasy-to-followstepsforconvenienceandspeed,whichcanbecompletedin3hoursfromDNAshearingtohMeDNAelution.The96stripwellmicroplateformatpermitsmanualhandlingbydoing1reactionatatimeorhighthroughputhandlingbydoing96reactionsatatime.HydroxymethylatedDNAfragmentsandnon-immuneIgGareincludedwiththekitasthepositivecontrolandnegativecontrol,respectively,forhydroxymethylatedDNAenrichmentfromanyspeciesincludingmammalian,plant,fungal,bacterial,andviraltypes.

SelectiveandHighEnrichment
TheuniqueprocedureandproprietarykitcompositionsallowforaselectiveandhighenrichmentofhydroxymethylatedDNAfromgenomicDNAsamplesforgene-specifichydroxymethylationanalysis.CombiningrealtimePCRwiththiskit,5-hmCenrichmentintheOCT4promoterregionwasdetectedtobe110timeshigherthanthatintheGAPDHpromoterregion(seeFig.2),indicatingthattheOCT4promoterishydroxymethylated.Thereisnocross-reactivitytomethylcytosineandunmethylatedcytosinesothatonlyhydroxymethylatedDNA(5-hmC)isenriched(seeFig.3).

CompatibleWithMeDIP
TheEpiQuik™hMeDIPkitcanbeusedwithMeDIPproceduresforasimultaneousanalysisofhydroxymethylatedandmethylatedstatusesofspecificgeneloci.

Responsive,Reliable,andPractical
Basedonitsworkingprincipleandthemicroplateformat,thekitcanbeusedinahighthroughputmannerwithDNAfromanyspeciesinavarietyofformsincludingculturedcells,freshandfrozentissues,paraffin-embeddedtissues,bloodsamples,andbodyfluidsamples.Todemonstratethecapabilitiesofthekit,ithasbeensuccessfullyusedforcapturinghydroxymethylatedDNAfractionfromgenomicDNAisolatedfromhumanbraintissues.ThepercentcontentofhydroxymethylatedDNAinhumanbrainsenrichedwiththekit(0.80%)iscomparabletothatmeasuredwiththe EpiQuik™HydroxymethylatedDNAQuantificationKit (0.67%),suggestingthatthehydroxymethylatedDNAfractionisfullycapturedwiththehMeDIPkit.

Fig.1.SchematicprocedureoftheEpiQuik™HydroxymethylatedDNAImmunoprecipitation(hMeDIP)Kit.

Fig.2. Sensitivedetectionofgene-specifichydroxymethylationbyhMeDIP-PCR |HumanbrainDNA(500ng)wasfragmentedto200-600bps.ThefragmentedDNAwasusedforhydroxymethylatedDNAenrichmentwiththeEpiQuik™hMeDIPKit.ElutedDNAwasanalyzedbyrealtimePCRwithprimersspecificallyforOCT4orGAPDHsequencesinthepromoterregions.ResultsshowthatthepromoterregionishydroxymethylatedinOCT4butnotinGAPDH.Fold-enrichmentrepresentstheamountofrecoveredDNAandwascalculatedbasedontheCtValue. 

 
Fig.3. SelectiveenrichmentofhydroxymethyatedDNAwiththeEpiQuik™hMeDIPKit |50pgofunmethylated,methylated,andhydroxymethylatedDNAcontrolwereeachspikedintofragmentedhumangenomicDNA(500ng).hMeDIPwasprocessedwiththe5-hmCantibodyandnon-immuneIgGincludedinthekit.ElutedDNAwasanalyzedbyrealtimePCRwiththecontrolprimersincludedinthekittodetectthepresenceofspikedcontrolDNA.Fold-enrichmentrepresentstheamountofrecoveredcontrolDNAandwascalculatedbasedontheCts.