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.