两种不同方法提取人粪便进行DNA检测的有效性研究

。1．SDBcl-2、BaxJ．2007226404－407．2CainKBrattonSBLanglaisCetal．Apaf-1oligomerizesintobiologi-callyactiveapproximately700-KDaandinactiveapproximately1．4-MDaapoptosomecomplexesJ．JBiolChem200027596067－6070．3PulsinelliWABrierleyJBPlumF．Temporalprofileofneuronaldam-ageinamodeloftransientforebrainischemiaJ．AnnNeurol1982115491－498．4．MorrisJ．200810855－57．5DasAGuytonMKMatzelleDDetal．Time-dependentincreasesinproteaseactivitiesforneuronalapoptosisinspinalcordsoflewisratsduringdevelopmentofacuteexperimentalautoimmuneencephalomye-litisJ．JNeurosciRes200886132992－3001．6HeAWangJAGuiCetal．Changesofmitochondrialpathwayinhy-poxiareoxygenationinducedcardiomyocytesapoptosisJ．FoliaHis-tochemCytobiol2007454397－400．7BrattonSBMacfarlaneMCainKetal．Proteincomplexesactivatedistinctcaspasecascadeindeathreceptorandstress-inducedapopto-sisJ．ExpCellRes2000256127－33．8ChengHYHsiehMTWuCRetal．Schizandrinprotectsprimaryculturesofratcorticalcellsfromglutamate-inducedexcitotoxicityJ．JPharmacolSci2008107121－31．9ShiY．ApoptosomethecellularenginefortheactivationofCaspase-9J．Structure2002103285－288．10AcehanDJiangXMorganDGetal．Three-dimensionalstructureoftheapoptosomeimplicationsforassemblyProcaspase-9bindingandactivationJ．MolCell200292423－432．11．、J．200474276－279．12LavrikINGolksAKrammerPH．Caspasespharmacologicalmanip-ulationofcelldeathJ．JClinInvest2005115102665－2672．13ZhuCWangXHagbergHetal．CorrelationbetweenCaspase-3ac-tivationandthreedifferentmarkersofDNAdamageinneonatalcere-bralhypoxia-ischemiaJ．JNeurochem2000752819－829．14EldadahBAFadenAI．CaspasepathwaysneuronalapoptosisandCNSinjuryJ．JNeurotrauma20001710811－829．［收稿日期2013－02－12］［本文编辑刘京虹吕文娟］·DNA何纯刚，黄沁园，陈利生，吴鸿根，邓洪强，潘云201012MS043Z20122465300215300215300211979－。[e-mail]。108DNA-DNADNA。-DNA232.51±41.24ng/μl、67.35±13.17ng/μlP＜0.01DNAOD260/280、-DNAP＜0.01。-DNAPCR5%1/2091.67%99/108P＜0.01。9DNADNA。DNADNA。-DNA。DNAR735A1674－3806201305－0408－04doi10．3969/j．issn．1674－3806．2013．05．03·804·ChineseJournalofNewClinicalMedicineMay2013Volume6Number5ResearchontheeffectivenessoftwomethodsforfecalDNAextractionanddetectionHEChun-gangHuangQin-yuanChenLi-shengetal．DepartmentofGeneralandPediatricSurgerythePeople’sHospitalofGuangxiZhuangAutonomousRegionNanning530021ChinaAbstractObjectiveToanalyzetheeffectivenessoftwomethodsforfecalDNAextractionanddetection．MethodsIn108patientswhowereinvestigatedfecalsampleswerecollected．ThefecalDNAwasextractedbyQIAampDNAStoolMiniKitandphenol/chloroformmethod．TheDNAqualityextractedbythetwomethodswascom-paredwitheachother．ResultsTheconcentrationsofDNAextractedbyphenol/chloroformmethodandkitwere232.51±41.24ng/μland67.35±13.17ng/μlrespectivelyP＜0.01．ThefecalDNAextractedbykitout-weighedthanphenol/chloroformmethodwhencomparedontheindexesofpurityOD260/280clarityofDNAsolu-tionandelectrophoresisresultsallP＜0.01．Therewere91.67%99/108samplesoffecalDNAextractedbykitcanbeamplifiedbyPCRwhileonly5%1/20samplesoffecalDNAextractedbyphenol/chloroformmethodcanbeamplifiedP＜0.01．TherewasnotenoughconcentrationofDNAgotfromanother8.33%9/108samplesofstoolatthefirsttimeextractedbykitbutcanachievetherequirementsbyimprovingthemethodatlast．ConclusionThefecalDNAmaybegothigherqualityextractedbykitwhencomparedwithphenol/chloroformmethodandcanachievetherequirementsofbisulfitemodificationandPCR．Improvingthephenol/chloroformmethodmayalsogethighquali-tativeDNA．KeywordsColorectalcancerEarlydiagnosisFecalDNAextraction1～3。DNADNA。DNADNA。11.1108612720。。61372416～8958。27151218～7854819＞2cm9144。2010325。1.21.2.110g1.5ml－80℃。1.2.2-DNA1201253180～220mg2mlTEbuffer600μl210%SDS200μl314000rpm1min2ml4K10mg/ml25μl55℃1h53min612000rpm5min1.5ml7、、25∶24∶112000rpm5min81/103MpH5.22.59101ml70%12000rpm5min11200μlTEbuffer。1.2.3DNA108DNADNA“QIAampDNAMiniKit”1200mg2ml21.6mlASL314000rpm3min41.4ml2ml51InhibitEX1min614000rpm3min71.5ml14000rpm3min8600μl2ml·904·201356525μlK9600μlAL15s1070℃10min11600μl12600μlQIAamp12000rpm1min213500μlAW1QIAamp12000rpm1min14500μlAW2QIAamp12000rpm1min15QIAamp2ml12000rpm1min16QIAamp1.5mlQIAamp200μlAE1min1712000rpm1minDNA－20℃。1.2.4DNA5μlDNA20OD260、OD280OD260DNAOD260/OD28ODNA。1.2.5DNADNA5μl1μl6×Loadingbuffer1.0%10V/cm30min。。DNA。1.2.6DNAPCR4p33ING1bDNAPCR。p33ING1bforward5-CTCGGGCCTATC-CACCTCTTCTG-3p33ING1breverse5-ACCAC-CACTCCCAGCAGCCTAG-3157bp。PCR25μlddH2O19.2μl10×LongTaqBuffer2.5μl2.5μmdNTPs1μlForwardprimer10μm0.5μlReverseprimer10μm0.5μlLongTaq2.5U/μl0.3μlDNA1μl。PCR94℃4min94℃30sec59℃45sec72℃30sec3572℃5min。PCR2.5%。1.3SPSS13.0±珋x±stχ2P＜0.05。22.1DNA、-DNA232.51±41.24ng/μlDNA67.35±13.17ng/μlt=17.744P＜0.01。DNADNA-DNA3/20χ2=105.860P＜0.01。-DNAOD260/2801.31±0.26DNAOD260/2801.84±0.12t=8.942P＜0.01。2.2DNA-DNADNA1203DNA。DNA91.67%99/108DNA、8.33%9/108DNA2。1-DNA2DNA2.3DNAPCR-DNA5%1/20DNA91.67%99/108P＜0.01。9400mgPCR3。·014·ChineseJournalofNewClinicalMedicineMay2013Volume6Number51～8PCR-DNA9～19PCRDNA3DNAPCR33.11010DNA。DNA5。DNA1～3DNA5～8DNADNA。DNA。3.2DNA-QIAampDNAStoolMiniKit200mgDNA。-DNADNAP＜0.01。DNADNADNA9。3.3DNA91.67%DNADNADNADNA。-DNADNADNADNA-DNA。3.4DNADNA、OD260/OD280-DNAP＜0.01。-DNA--。DNADNA。3.5DNAPCRPCRp33ING1b-DNA1/20PCRDNAPCR9PCR。DNAP