The EpiQuik™CirculatingCell-FreeDNA(cfDNA)IsolationEasyKit utilizesmagneticbeadsbasedsize-fractionationtechnologytoisolatecirculatingcell-freeDNA(cfDNA)fromplasma/serumsamplesinasimpleandfastmanner.TheisolatedcfDNAcanbedirectlyusedforrealtime-PCRandDNAlibrarypreparationsuitablefornextgenerationsequencing. Thekithasthefollowingadvantages: - Fastandstraightforwardprocedurecanbefinishedwithin1hour.
- Usesinnovativemagneticbeadbasedsize-fractionationtechnologyforisolationofcfDNAfromplasma/seruminasimpleandconvenientmanner.
- TheisolatedDNAcanbedirectlyusedforbothqPCRandNGSDNAlibrarypreparation.
- Efficientremovalofproteins,salts,nucleases,PCRinhibitingsubstances,andotherimpuritiessuchaspolysaccharides,polyphenolsandlipids.
- SensitiveandefficientDNAcaptureenablessuccessfulisolationwithhighrecovery(>80%ofinputmononucleosomalDNA),evenwhenthequantitiesofstartingmaterialarelimited(aslowas0.2ml).
BackgroundInformation
Geneticandepigeneticanalysisofcirculatingcell-freeDNA(cfDNA)inplasma/serumorotherbodyfluidsprovidesuniqueopportunitiesforearlydetectionofawiderangeofclinicaldisorderssuchascancer,autoimmunedisease,infectionandfetaldisorders.ItwasdemonstratedthatcfDNAofclinicalimportanceoccurspredominantlyasfragmentsofapproximately170basesfrommononucleosomeswithasmallerproportionasfragmentsof360basesfromdi-nucleosomes[1,2].Suchnucleosomalcomplexesarereleasedintobloodcirculationduringapoptoticcelldeathandwillbeincreasedundervariouspathologicalcircumstancessuchasinflammation,pulmonaryembolism,autoimmunedisease,andcancer[3,4].ItisalsoshownthatcfDNAfromnucleosomalcomplexesinserumandplasmaissmallsizefragmentDNA(170-500bps)andusingsuchcfDNAforgeneticorepigeneticanalysisprovidesbetterandmoreaccurateidentificationofphysiologicalandpathologicalstatus[5]. Principle&Procedure
TheEpiQuik™CirculatingCell-FreeDNA(cfDNA)IsolationEasyKitcontainsallcomponentswhichhavebeenoptimizedforthesimpleandrapidisolationofsmallsizecfDNAfromplasma/serum.ThecirculatingnucleosomalcomplexesarefirstdigestedandDNAisthenenzymaticallyreleased.ThecfDNAisefficientlycapturedviasize-fractionationmagneticbeads(cfDNACaptureBeads)byapplyingthebeadstoamagneticfield (EpiMag™HT(96-Well)MagneticSeparator orsimilar).ThecapturedcfDNAispurifiedbysimplywashingthe beads.ThepurifiedcfDNAisthenelutedfromthebeadsforimmediateuseorstorage. StartingMaterials
Bothfreshandfrozenplasma/serumfromvarioussourcescanbeused.However,freshplasma/serumwillgenerallygivehigherDNAyieldsthanfrozen.Theinputvolumeofplasma/serumcanbefrom0.1-1mlwiththestandardvolumeof0.5mlpersample.Ifserumsampleisused,theserumshouldbepreparedwithin6hoursafterblooddraw,sincelysisofperipheralbloodlymphocytesmaycauseanartificialincreaseintheamountofDNAduringserumseparation.
References: 1.JahrSetal:CancerRes.2001,61:1659-1665
2.SuzukiNetal:ClinChimActa.2008,387:55-58
3.HoldenriederSetal:CritRevClinLabSci.2009,46:1-24
4.SchwarzenbachHetal:NatRevCancer.2011,11:426-437
5.ChanKCAetal:ClinicalChem.2004,50:88-92 | 
 Fig.1. WorkflowofEpiQuik™CirculatingCell-FreeDNA(cfDNA)IsolationEasyKit.
Fig.2. HighrecoveryofcfDNA:DifferentamountsofHeLamononucleosomeswerespikedinto0.5mlofplasmathenisolatedusingtheEpiQuik™CirculatingCell-FreeDNA(cfDNA)IsolationEasyKit.TheisolatedDNAwasfluorescentlyquantified. 
Fig3. HighrecoveryofcfDNAconfirmedbybioanalyzeranalysis: 500ngofHelapolynucleosome(around3000bps)and500ngofmononucleosome(around170bps)weresimultaneouslyspikedinto0.5mlplasma andthenisolatedandsize-selected. 
Fig4. BioanalyzertraceofDNAlibrarypreparedfrom20ngofpurifiedmononucleosomesisolatedfromplasmaspikedwithbothmono-andpoly-nucleosomes.Librarypeaksize:350bps. | ProductComponents
ProteinaseK
cfDNACaptureEnhancer
cfDNACaptureBeads
CaptureBuffer
ElutionBuffer
UserGuide
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