23423.selexseq繪制nfkbdna結(jié)合譜

1、東南大學(xué)碩士學(xué)位論文SELEXSeq繪制NFkBDNA結(jié)合譜姓名：陸潔申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：生物醫(yī)學(xué)工程；生物物理指導(dǎo)教師：王進(jìn)科201105AbstractTitle：ProfilingDNAbindingspectrumofNFcBwithSELEXSeqM漁STER：LUJieSUPERVISORS：ⅥrANGJinkeSchool：SoutheastUniversityDNAbindingspectrumoftranscrip

2、tionfactorreferstoallDNAsequenceswhichcanbeboundbytranscriptionfactorinaspecificmannerandtherelativebindingaffinityoftranscriptionfactortothemTheprofilingofDNA—bindingspectrumisofgreatvalueinresearchingDNAbindingspecific

3、ityandforecastingDNAbindingtargetsoftranscriptionfactorsAtpresent，anewtechniquenamedSELEXSeqwhichcombinesthetraditionalSystematicEvolutionofLigandsbyEXponentialenrichment(SELEX)withadvancedDNAparallelsequencingtechnology

4、becamethebestandmostadvancedmethodforprofilingDNAbingdingspectrumoftranscriptionfactonNuclearfactorrB(NF一1cB)isasequencespecificDNAbindingtranscriptionfactorwhichplayscriticalrolesincellilnnluneresponse，inflammatoryreact

5、ionandcancerdevelopmentAtpresent，NF心hasalreadybecomeallimportanttargetfordrugscreeningTherefore，inordertofullyunderstandtheDNAcombiningspecificityofNFrBandtopredictitsglobalDNAtargets，thispaperemployedSELEX—Seqtechniquet

6、oprofilethelligh—resolutionDNAbindingspectrumofNFrBp50protein1rtlemainexperimentalstepsareasfellows：first，10一ntsingle—strandedDNA(ssDNA)librarypoolWasdesignedandchemicallysynthesisedbasedonthelengthoftheknownNF—rBDNAbind

7、ingsite(forexample，GGGACTTTCC)SecondlyssDNAlibrarywasconvertedintodouble—strandedDNA(dsDNA)librarybyannealingaprimerandextendedwithDNApolymeraseTheprepareddsDNAlibrarypoolwasthenperformedSELEXscreeningbyusingEMSA(Electro

8、phoresisMobilityS1liRAssay)AfterfourroundsSELEXscreeningthedsDNAswhichboundbyNF一1(Bp50proteinwereenrichedTheenricheddsDNAwasfinallysequencedwithSolexasequencingtechnologyand9667032readswasyieldAftercleaningrawdatabyremov

9、ingreadswithwrongPCRprimerandT5sequence，7292238readswereobtainedandappliedtobioinformaticsanalysisThroughseveralanalysesofmanysampledreadswithMEMEprogramthesimilarDNAbindingmotifsofNFrBp50wereobtainedTherelativebindingaf

10、finityofNF—rBp50toallreadsWascalculatedbycountingtheirnumbersinallsequencedreadsandconfirmedbydetectingthebindingofNFrBp50tosometypicalreadswithvariousbindingaffinitywithanearinfraredfluorescenceEMSAexperimentTllispapere