《III提取DNA》PPT课件.ppt

Chapter3,DNA的提取与纯化,基因工程需要三种不同的DNA:总DNA质粒DNA噬菌体DNA,Figure3.1ThebasicstepsinpreparationoftotalcellDNAfromacultureofbacteria,BasicSteps,1总DNA提取,1.1培养、收集细菌细胞,两种类型的细菌培养基的成分：M9培养基：Na2HPO4(6.0)KH2PO4(3.0)NaCl(0.5)NH4Cl(1.0)MgSO4(0.5)Glucose(2.0)CaCl2(0.015)LB培养基：typtone(10)Yeastextract(5)NaCl(10),1.1培养、收集细菌细胞,37C，150-250rpm达到最大浓度2-3109cell/ml。600nm下的OD值，1OD相当于0.8109cell/ml,Figure3.2Estimationofbacterialcellnumberbymeasurementofopticaldensity,Figure3.3Harvestingbacteriabycentrifugation,Figure3.4Preparationofacellextract.,Preparationofacellextract,1.2裂解细胞,溶菌酶、EDTA,Figure3.5Removalofproteincontaminantsbyphenolextraction.,3PurificationofDNAfromacellextract,1.4DNA的浓缩与浓度测定,最常用的方法是乙醇（同时含有Na离子）、异丙醇沉淀法。260nm下测定吸光度，1OD相当于50g双链DNA/ml。A260/A280=1.8，1.8说明有RNA。,Figure3.6CollectingDNAbyethanolprecipitation.,ConcentrationofDNAsamples,Figure3.7TheCTABmethodforpurificationofplantDNA,PlantDNA(CTAB法),十六烷基三甲基溴化铵,Figure3.8Theuseofananion-exchangechromatographyresininDNApurification.,阴离子交换层析法,2质粒DNA提取,质粒DNA的大小（8%）。DNA的构造AlkalinedenaturationEthidiumbromide-caesiumchloridedensitygradientcentrifugation,sphaeroplasts裂解细胞可以破坏部分细胞壁，而保持细胞膜的完整。问题：裂解液中包含染色体DNA质粒分子非常大时，将与染色体DNA共沉淀,Figure3.9Preparationofaclearedlysate.,Sphaeroplast残壁细胞,碱裂解法基本原理：在pH12.012.5范围内，非螺旋化的DNA会变性，而闭合环状的质粒的DNA则不会被变性或只有少量的氢键断裂；当恢复中性pH值时便会迅速地复性。但变性的线性染色体DNA分子复性时不准确，也不迅速，因此彼此聚集形成网状结构，通过离心分离便与变性的蛋白质及RNA一起沉淀下来，而仍滞留在上清液中的质粒DNA则可用酒精等沉淀收集。,Figure3.11Plasmidpurificationbythealkalinedenaturationmethod.,Alkalinedenaturation,Figure3.10Twoconformationofcirculardouble-strandedDNA.,Twoconformationofcirculardouble-strandedDNA,CircularformsofDNAmigrateinagarosedistinctlydifferentlyfromlinearDNAsofthesamemass.Typicallyuncutplasmidswillappeartomigratemorerapidlythanthesameplasmidwhenlinearized.Additionally,mostpreparationsofuncutplasmidcontainatleasttwotopologically-differentformsofDNA,correspondingtosupercoiledformsandnickedcircles.Theimagetotherightshowsanethidium-stainedgelwithuncutplasmidintheleftlaneandthesameplasmidlinearizedatasinglesiteintherightlane.,Figure3.12CsCldensitygradientcentrifugation.,CsCldensitygradientcentrifugation,EBCsCl密度梯度离心法,EB插入相邻的碱基之间，降低了DNA的浮力密度，但超螺旋的DNA结合EB浮力密度降低较小，仅有0.085g/cm3。,Figure3.13PartialunwindingoftheDNAdoublehelixbyEtBrintercalationbetweenadjacentbaseparirs.,EB,EB如何与DNA结合?,Figure3.14PurificationofplasmiodDNAbyEtBr-CsCldensitygradientcentrifugation,如何分离质粒DNA？,2.3质粒扩增,一些多拷贝质粒具有在无蛋白合成条件下复制的能力。加入蛋白合成抑制剂如氯霉素，可以获得上千个拷贝。,Figure3.15Plasmidamplification,Plasmidamplification,Inhibitorofproteinsynthesis:Chloramphenical,12h,3噬菌体DNA的制备,当培养物离心时，细菌沉淀到底部，噬菌体留在悬浮液中，去蛋白就可以获得噬菌体DNA。每ml菌液可以获得1010噬菌体，但只能产生500ngDNA。,Thelysogenicinfectioncycleofbacteriophage,3.1噬菌体的培养,cI突变的温度敏感突变体。,Figure3.17Inductionofacltslysogenbytransferringfrom30to42,Lysogenicphage,At30,cltsgeneproteinworks,Keeplysogeny:At42,cltsgeneproteindoesnotwork,produceextracellularphage.,Figure3.18Achievingtherightbalancebetweencultureageandinoculumsizewhenpreparingasampleofanon-lysogenicphage,Non-lysogenicphage（Lytic),由于缺失修饰，大多数基因工程载体属于此类型。,3噬菌体DNA的制备,3.2收集噬菌体噬菌体颗粒非常小，需高速离心沉淀。PEG可使病毒颗粒形成多聚体。较低离心力即可沉淀噬菌体。,3噬菌体DNA的制备,3.3从噬菌体中纯化DNAPEG沉淀物中可能含有少量的细菌裂解物，也可能含有细菌DNA。可通过CsCl梯度离心去掉。通过酚抽提或者蛋白酶处理蛋白质外壳。,Figure3.19Collectionofphageparticlesbypolyethyleneglycol(PEG)precipitation,Collectionofphageparticles,Purificationofphage,Figure3.20PurificationofphageparticlesbyCsCldensitygradientcentrifugation.,4纯化M13DNA,每ml菌液可以获得1012的噬菌体。少量的培养物可以获得大量的噬菌体。细菌细胞不裂解，不存在细胞裂解物的问题，也不需要CsCl梯度离心