Wholegenomeamplification(WGA)withlessbiasmakingthiskitideallysuitedfornextgenerationsequencingapplications.
- Highsensitivity:Typicalyieldisgreaterthan5µgwhenstartingwith1ngofgenomicDNA.
- Reducedamplificationbiasandnoprimerartefacts:Primer-freeamplificationmethodutilizesacombinationofTthPrimPolPrimase(tosynthesizeprimers)andPhi29DNApolymerase.
- Wellsuitedfornextgenerationsequencing:TestedinIlluminaandIonTorrentNGSworkflows.
- Easyandreliable
- InsensitivetoexternalDNAcontamination
TheTruePrime™WGAKitusesarevolutionarymultipledisplacementamplificationmethodbasedonthecombinationoftherecentlydiscoveredDNAprimase“TthPrimPol”andtheextremelyprocessiveandhigh-fidelityPhi29DNApolymerasetouniformlyamplifygenomicDNAfrompurifiedstartingmaterial.TheextraordinarystranddisplacementcapacityofthePhi29DNApolymeraseallowsTthPrimPoltogeneratenewprimersonthedisplacedstrandsthatareextendedbyPhi29DNApol,resultinginexponentialisothermalDNAamplification.ThetypicalDNAyieldis>5µgfromasinglereactionusing1ngofstartingDNA.
- HowdoesTruePrime™Technologywork?
- Wholegenomeamplificationfrom6pgofhumanDNA
- Absenceofprimerartefacts
- InsensitivitytoexternalDNAcontaminants
- Excellentgenomecoverage
- Extremesensitivity
- Highyields
HowdoesTruePrime™technologywork?
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ExamplesofTruePrime™wholegenomeamplification
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Absenceofprimerartefacts
1pgofhumangenomicDNA(~1/6ofthecontentofonehuman/mammaliancell)hasbeenamplifiedusingeitherTruePrime™(TthPrimPol-basedMDA)orrandomprimedMDAreactions.Randomprimedreactionscontain20%ofsequencesthatcannotbemappedtoanyorganisminsequencedatabases.
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InsensitivitytoexternalDNAcontaminations
Duetothehighsensitivityofsinglecellwholegenomeamplificationtechniquesthereisaconsiderabledangerofcontaminatingreactionswithnon-targetderivedDNA.Wethereforerecommendtotakeextremecarewhensettingupsinglecellamplificationreactionsaslaidoutinthehandbook.However,TruePrime™usershaveauniqueadvantagehere:TruePrime™doesnotacceptnon-denaturedDNAstrandsastemplateasreADIlyasrandomprimedMDAreactions.Therefore,contaminationscominginfromtheenvironmentetc.afterthedenaturationstepwillnothaveastronginfluenceonthecompositionofyourreactionoutput.WehaveshownthisbehaviourinanexperimentwherewesimulateanexternalDNAcontaminationbydeliberatelyaddinginyeastDNAtoadenaturedhumanDNAtemplate.YeastDNAisinathousand-foldexcessoverthetargetDNA(1pghumangenomicDNA):
WhereaswithTruePrime™thevastmajorityofthesequencesobtainedaretarget-derived,randomprimedMDAshowsamajorityofcontaminant-derivedsequences.
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Excellentgenomecoverage
WehaveamplifiedandsequencedtheyeastgenomeusingTruePrime™andIlluminatechnology.Using2.5millionreadpairswecover99.4%ofthetotalyeastgenome.Shownisthecoveragewithnarrowwidthofthedistributioncurve.
Acoveragegraphofyeastchromosome7exemplifiesthemoreevencoverageobtainedbyTruePrime™MDA(middlebluelinerepresentsmeancoverage).
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TruePrimeshowsanextremesensitivity
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TruePrime™produceshighyieldsofDNAfrom1fgofinput.
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ORDERINFORMATION
EachTruePrime™WGAKitcontains:BufferD,BufferN,ReactionBuffer,dNTPs,Water,Enzyme1andEnzyme2.ThecompletemanualisavailableundertheManualtab.LucigenisanauthorizeddistributorofSygnisproductsintheUS.
TruePrime™WGAKitisintendedformolecularBIOLOGyuseonlyandinvitrouseonly.Thisproductisnotintendedfordiagnosis,preventionortreatmentofadiseaseinhumanbeingsoranimals.
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