TheMethylamp™MS-qPCRFastKitisacompletesetofessentialcomponents,whichenablestheexperimentertoperformfast,specific,sensitiveandreproducIBLemethylation-specificquantitativePCR.TheMethylamp™MS-qPCRFastKitisverysuitableforquantitativemethylation-specificPCRinafastformatusingveryminuteamountsofDNA.ThenovelhotstartDNApolymerasecontainedinthiskitcanspecificallyreducetheoveralltimerequiredforMS-qPCRfromapproximately2.5hourstolessthan70minutes.ThekitalsofacilitatessensitivityandspecificityofMS-qPCRbysignificantlyincreasingprimer-bisulfiteDNAtemplateannealing,whilesimultaneouslyreducingnon-specificannealing.ThisresultsinconsiderabletimesavingsandamoreefficientMS-qPCR.Thekithasthefollowingadvantages:

• Anextremelyfastprocedurethatcanbefinishedwithin70minutes.
• Abundantyieldsduetohighamplificationefficiency.
• HighlyaccurateandspecificinMSP,withreducedfalse-positiveresults.
• Convenientmastermixformattoalloweasyreactionsetup.
• Simple,reliable,andconsistentassayconditions.
• Canbeusedwithanyblock-basedreal-timePCRinstrument.

Principle&Procedure
TheMethylamp™MS-qPCRFastKitprovidesamastermixformat,whichcontainsahotstartDNApolymerase,dNTPs,anMS-PCRenhancer,anoptimizedbuffer,andanintercalatinggreendye.Thismastermixallowsforaconvenientandeasyreactionsetup.TheuniquehotstartDNApolymeraseisprovidedinaninactivestateatambienttemperatureandisreactivatedbyseveralminuteincubationsat95°C,whichcaneasilybeintegratedintoexistingthermalcyclingsteps.ThehotstartDNApolymeraseincombinationwiththeoptimizedbufferensurestheMS-qPCRspecificityandsensitivity.ThegreendyeallowsforDNAdetectionandanalysiswithoutusingasequence-specificprobe.

 
Fig.1.SchematicprocedureforusingtheMethylampMS-qPCRFastKit.

Fig.2. MS-PCR amplificationofageneregionineuchromatinwithinternalcontrolprimers.SuccessfulGpCmethylationandbisulfiteconversionwerecarriedoutusingan EpiQuikChromatinAccessibility&MethylomeSequencing(CAMe-Seq)Kit(Cat.No.P-1048)andPCRwasperformedusingMethylamp™MS-qPCRFastKit(Cat.No.P-1028).