9.-Introduction-to-technology-of-molecular-biology.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,9.1 Gene manipulation,9.2 Clone vectors,9.3 Gene libraries and screening,9.4 Analysis and uses of cloned DNA,9.Introduction to technology of molecular biology,9.1 Gene manipulation,9.1.1 DNA cloning:an overview,9.1.2 Preparation of plasmid DNA,9.1.3 Restriction enzymes and electrophoresis,9.1.4 Ligation,transformation and analysis of recombinants,9.1.1 DNA cloning:an overview,DNA cloning,.DNA cloning facilitates the isolation and manipulation of fragments of an organisms genome by replicating them independently as part of an autonomous vector.,Hosts and vectors,.,E.coli,as the host organism,plasmids and bacteriophages,as cloning vectors in,E.coli,.(Ti plasmid in plant genome;M13,;plasmid-,=cosmid,),Vectors,based on plasmids,viruses and whole chromosomes have been used to carry foreign genes into other prokaryotic and eukaryotic organisms.,Subcloning,is the simple transfer of a cloned fragment of DNA from one vector to,another,which examplifies many of the basic techniques of DNA cloning.,Steps:,Isolation,of plasmid DNA,containing interest sequence,Digestion,(,cutting)the plasmid into fragments by RE,Separation,by,agarose,gel electrophoresis,Purification,of target fragments,Ligation,(,joining)of the fragment into a new vector,Selection,of transformed bacteria,Analysis,of recombinant plasmids,DNA,libraries,DNA Libraries,are sets of random cloned fragments of either genomic DNA of cDNA,each in a separate vector molecule,are used in the isolation of unknown genes.,There are two main sources been designed to identify an unknown gene-bulk genomic DNA,bulk mRNA,they are used in formation of,genomic libararies,and,cDNA libararies,.,Genomic,libraries may be an inefficient method of find a gene,where much of DNA is,noncoding,.,cDNA,are synthesized from the mRNA by reverse transcription and are then insert into a vector to form a,cDNA,library,.,Screening Libraries,Libraries,are screened for the presence of,a gene sequence,by,hybridization,with a sequence,(a radioactively or fluorescently labeled DNA probe)derived from its protein product or a related gene,or through the screening of the protein products of the cloned fragments.,Alalysis,of a clone.,Once,indentified,a cloned gene may be analyzed by,restriction mapping,and ultimately by,DNA sequencing,before being used in any of the diverse applications of DNA,cloning.,Many enzymes are used,in vitro,in DNA cloning(p108 table 1).,9.1.2 Preparation of plasmid DNA,Plasmids as vectors,.,Bacterial plasmids,small circular DNA molecules,which replicate independently of the genome and encode aitibiotic resistance(bla,amp,r,),are,the commonest vectors,for carring cloning DNA.,Phenol extraction,酚提,Extraction with,phenol,or phenol-chlroform,misture,removes any remaining proteins,from an alkaline lysate.,Phenol-,剧烈振荡可与水混溶后分离，沉淀蛋白于界面间,。,Ethanol precipitation.,乙醇沉淀,The DNA and RNA remaining in the aqueous layer are now,concetrated,by ethanol precipitation.,Cedium,chloride gradient,核酸的分离与提纯,原理：DNP（染色体DNA与组蛋白结合成的核蛋白）溶于水合浓盐溶液（如1 mol/L NaCl），但不溶于生理盐溶液（0.14 mol/L NaCl）。通过此性质纯化核蛋白。,苯酚：,强蛋白质变性剂。水饱和的苯酚与DNP一起振荡，冷冻离心，DNA溶于上层水相，不溶性变性蛋白质残留于中间界面，一部分变性蛋白质停留在酚相。将含DNA水相合并，在有盐存在的条件下加2倍体积冷乙醇，可将DNA沉淀出来。,用乙醚和乙醇洗沉淀。可得到纯DNA。（用氯仿异戊醇与DNP溶液振荡，借助表面变性除去蛋白质，反复多次直至得到不含蛋白质的DNA。与苯酚法相似。）,在细胞悬浮液中直接加入2倍体积的1十二烷基硫酸钠（SDS）缓冲溶液（避免核酸酶和机械振荡对DNA的降解），以及光谱性蛋白酶，如蛋白酶K（可使细胞内蛋白质全部降解）以得到大分子DNA,。,Phenol extraction,.,核酸的超速离心,密度梯度沉降平衡超速离心法应用：测定核酸的密度；测定DNA中G-C含量；溶液中核酸构象研究；核酸制备。,Cesium chloride gradient.,9.1.3 Restriction enzymes and electrophoresis,Restriction,endonucleases,Restriction endonucleases are,bacterial enzymes which,cut,DNA into defined and reproducible,fragments,.,They,are the basic tools of,gene clonging.,They consist of two components:,the first is,restriction endonucleases,;,the second is,methylase,(,甲基化酶,),adds a methyl group to a C or A base within the same recognition sequneces in the cellular DNA.,Recognition,sequences,Restrictions enzymes cleave DNA,symmetrically,(,对称地,)in both strands at,short palindromic(,回文,)recognition sequences,to leave a 5,-phosphate and a 3-OH.,They leave blunt(,钝,)ends,or protruding(,凸,)5-or 3termini.,Cohesive,ends,Restrictions enzyme products with single-stranded termini are said to have,cohesive ends or sticky ends,since they can anneal(,退火,)by base pairing to any other fragments with a complementary terminus.,Restriction,digests,限制性酶解,Commercially supplied enzymes,(,商品酶,)are used to digest plasmid DNA before analysis or purification of the fragments by agarose gel electrophoresis.,（,p115 fig.2,）,Agrose gel electrophoresis,Agarose gels,separate linear DNA,on the basis of,size,by the,migration,(,迁移,)of DNA through a matrix(,模具,)under the influence of an electric field.,Electrophoresis may be used to,determine the gross organization,(,大致构型,)of plasmid molecules.,Isolation of fragments,Specific DNA fragments may be cut out of agrose gels and purified for use in subsequent cloning experiments.,（,p116 fig.3,）,;,（,p117 fig.4,）,gel electrophoresis,凝,胶,电泳,（,1,）,Agarose,gel electrophoresis,琼,脂糖凝胶,电泳（,0.2-50kb,）,迁,移率决定于以下因素,：,核酸分子大小,；,凝胶浓度,；,DNA,的构象；,电压,；,碱基组成,；,温度,；,电流量,。,溴化乙锭（,EB,）染色：电泳后的凝胶需在,EB,的水溶液中染色。,凝胶上的样品可回收供进一步研究用,。,（,2,）,聚丙烯酰氨凝胶,电泳（,1-1000bp,）,凝胶孔径小，可分析相对分子量较小的,DNA,或,RNA,片段。凝胶强度较好，通常用垂直板电,泳。,（,3,）脉冲电场凝胶电泳（,50kb-10000kb,）,9.1.4 Ligation,transformation and analysis of recombinants,DNA ligation,T4 DNA ligase,repairs,breaks in a dsDNA backbone and,can covalently rejoin,annealed(,退火,)cohesive ends in the reverse of a restriction enzyme reaction,to create new DNA molecules.,Recombinant DNA molecules,Alkaline phosphatase,碱性磷酸酶,Treatment of the linear vector,molecule with,alkaline phosphatase,will,remove the 5,phosphates,and,render the vector unable to ligate into a circle,without an inserted target,so reducing the proportion of recreated vector to the mixture.,Transformation,Transfomation,is the process of take-up of foreign DNA,normally plasmids,by bacteria.,Plasmids are cloned by tranfer,into strains,of,E.coli,with defined genetic properties.,The,E.coli,cells can be made,competent,(,易接受质粒的，感受态的,)to take up plasmid DNA by treament with Ca,2+,.,The cells are,plated out on agar,and grown to yield,single colonies,or clones,.,Selection,By growth on a plate containing an antibiotic,Transformation efficency,The number of antibiotic-resistant colonies per microgram of input plasmid DNA.,Screening transformants,Many case vectors designed,Subcloning experiment digest the DNA from miniprep,gel.,Growth and storage of transformants,Single colonies are grown in liquid medium,mainatinng the antibiotic selection for the plasmid,and a portion of the culture is stored for later use as a,frozen glycerol,stock.,Gel analysis,Recombinant plasmids can be,distinguished from vectors by size on an agarose gel,and by,excising the inserted fragment with the same restriction enzymes used to insert it,.,Fragment orientation,The orientation of the insert in the vector may be determined using an,agarose gel,by,digestion of the plasmid with a restriction enzyme known to cut asymmetrically,不对称,within the insert sequence,.,聚合酶链式反应（PCR）,（1）基本原理,模仿体内DNA复制过程，首先使DNA变性，解链；然后使引物与模板退火，二者碱基配对。DNA聚合酶即以4种dNTP为底物，在引物的引导下合成与模板互补的DNA新链。重复此过程，DNA链以指数方式扩增。最初采用的聚合酶是klenow片段，1988年后分离出耐热的Taq DNA聚合酶。扩增公式：Y=(1+X)n Y-产量、X-扩增效率、n-循环次数。,（2）基本步骤：,设计一对引物；优化反应体系；适量模板、引物、4种dNTP、Taq DNA聚合酶、Mg2+；选择3个循环温度，变性94，45-60s；退火两个引物中较低的Tm值减2；1min；延伸72，1min；取出反应物，检测扩增结果。,（3）发展与应用：,合成特异探针；DNA测序；RT-PCR；产生和分析基因突变；重组PCR；未知序列PCR扩增；基因组序列的比较研究；临床医学和法医学中的应用。,（4）实时定量PCR(real time quantitative PCR)技术,利用带荧光检测的PCR仪对整个PCR过程中扩增DNA的累积速率绘制动态变化图，从而消除了在测定终端产物丰度时有较大变异系数的问题。,荧光探针：仅能与目的DNA序列特异性结合。,思考题,DNA cloning,vectors,Subcloning,DNA Libraries,cDNA Libraries,plasmid,miniprep,Alkaline lysis,Phenol extraction,Restriction endonucleases,T4 DNA ligase,Alkaline phosphatase,Transfomation,competent cell,Transformation efficency.,简述,subcloning,基本步骤。,简述质粒,DNA,制备基本过程。,常见凝胶电泳包括哪三种？电泳迁移率影响因素有哪些？,叙述,PCR,的基本原理和步骤。,Thank you for your attention!,