1、第二章 分子生物学方法Chapter 2 Methods of Molecular Biology,Methods of Molecular Biology,2.1 Molecular separations 分子的分离2.2 Gene cloning techniques基因克隆技术2.3 Labeled tracers（标签追踪）2.4 Hybridization（杂交）,Restriction Endonuclease(限制性核酸内切酶)Vector（载体）Plasmids（质粒）Phages（噬菌体）Cosmids（黏粒）M13 Phage vectorsPhagemids（噬菌粒）E
2、ukaryotic Vectors and very high capacity vectors（真核生物载体和高通量载体）Expression Vectors（表达载体）Inducible Expression Vectors（可诱导的表达载体）Shuttle vector（穿梭载体）PCRcDNA CloningRACEDNA sequencing,小结 第二章 作业,2.1 Molecular Separations（分子的分离）,Agarose gel electrophores is（琼脂糖凝胶电泳)pulsed-field（脉冲场凝胶电泳）Polyacrylamide gel el
3、ectrophoresis（聚丙烯凝胶电泳）Two-dimensional polyacrylamide gel electrophoresis（双向聚丙烯凝胶电泳）Ion-exchange chromatography（离子交换层析）Gel filtration chromatography（胶体过滤层析）Affinity chromatography(亲和层析),Separate proteins/nucleic acid（分离蛋白/核酸）Purify a particular proteins/nucleic acid（纯化一个特殊的蛋白/核酸）,聚丙烯凝胶电泳分辨率高，适合分辨较小的D
4、NA或蛋白质；琼脂糖凝胶电泳分辨率较低，适合分离较大的核酸；极长的DNA（3050kb）需要用脉冲场凝胶电泳；,DNA gel electrophoresis,DNA带负电,See page 652,怎样根据标准分子量换算目的大DNA的分子量？DNA的迁移率与其片断大小的对数成正比。大小在0.21.2kb之间的双链DNA片段的凝胶电泳试验结果。在该图的基础上回答以下几个问题：a.迁移率为16mm片断的大小为多少？b.大小为0.5kb的片断的迁移率为多少？,脉冲场（pulsed-field）凝胶电泳,See page 653,酵母染色体脉冲场凝胶电泳,SDS-polyacrylamide gel
5、 electrophoresis,Protein gel electrophoresis,聚丙烯凝胶电泳能否分离核酸？为什么？,常用蛋白质分子量标准参照物,用双向电泳和质谱分析进行蛋白质组分析,See page 683,根据荷质比,用胰蛋白酶处理，在每个带正电荷氨基酸后进行切割多肽链（K：赖氨酸；R：精氨酸）,Two-dimensional gel electrophoresis,Separate all of the thousands of polypeptides present at a given time in a given cell type.,Proteomics 蛋白质组学
6、,Isoelectric focusing,SDS-PAGE,Ion-exchange chromatography（离子交换层析）,Separating substances according to their charges根据物质的电荷进行分离例1,DEAE-Sephadex(交联葡聚糖)chromatography with positive charges（正电荷）Phosphocellulose(磷酸纤维素)with negative charge（负电荷）,Ion exchange chromatographyTwo forms of an enzyme are separat
7、ed,See page 680,Multiple forms of eukaryotic RNA polymerase真核生物的多种RNA聚合酶的分离,Sea urchin embryos（海胆胚胎）DEAE-Sephadex ion-exchange chromatographyGreen,protein contentRed,RNA polymerase activity,RNA polymerase IRNA polymerase IIRNA polymerase III,Gel filtration chromatography,Separating substances accord
8、ing to molecular size,Gel filtration resins are porous beads,See page 680,Affinity chromatography(亲和层析),亲和层析是一种吸附层析，免疫亲和层析（immunoaffinity chromatography）是最常见的一种蛋白质亲和层析。珠子上结合了一种靶蛋白（抗原）的特意抗体。理想的抗体只与靶蛋白发生特异性结合（而这种结合在一定的条件下又是可逆的）。其他蛋白质都流出柱子。所以将抗原（或抗体）固相化后，就可以使存在液相中的相应抗体（或抗原）选择性地结合在固相载体上，借以与液相中的其他蛋白质分开，达
9、到分离提纯的目的。添加组氨酸标签,See page 680,2.2 Molecular Cloning Methods（分子克隆方法）,Gene Cloning（基因克隆）,DNA克隆：构建重组DNA分子并在细胞中保存和扩增这些分子，叫DNA 克隆；Cloning gene by cloning its bacterial host.基因是被他的细菌寄住克隆的。,Recombinant DNA technique(重组DNA技术)molecular cloning（分子克隆）gene engineering（基因工程）gene cloning（基因克隆）,真核基因被连接到小的细菌或噬菌体DNA
10、和将这些重组分子插入到细菌寄住中，产生大量相同的基因。,第一个试管中得到的重组DNA克隆,常用到的名词,p658,See Molecular Biology page 61,The first cloning experiment involving a recombinant DNA assembled in vitro.这是第一个试管中得到的重组DNA克隆,氨苄青霉素(Ampicillin).卡那霉素(Kanamycin).四环素（Tetracycline）.氯霉素(Chloramphenicol)链霉素（Streptomycin）.潮霉素（Hygromycin）,常用的选择标记有：,Cu
11、tting 切割 Linkage 连接 Replication复制 Transfer 转移 Restriction endonuclease（RE）限制性内切酶 Nuclease 核酸酶 Ligase 连接酶 Polymerase 聚合酶 Phosphatase and kinase 磷酸化酶和激酶 Methylase甲基化酶,常用到的名词：,See page 654,2.1.1.限制性内切核酸酶Restriction Endonuclease(RE,Restriction enzyme)比喻：切DNA分子的小刀（DNA molecular knife to cut DNA）限制性内切核酸酶是
12、一类识别特定的DNA序列并在特定位点切割DNA的核酸水解酶“Restriction enzyme recognize specific short sequences of DNA and cleave the duplex(sometimes at target site,sometimes elsewhere,depending on type)”.,See page 655-656,平头末端,粘性末端,3黏性末端,回文序列,一个EcoRI位点的切割EcoR在两条链的识别位点内切割，从而产生5突出末端。,5黏性末端,计算题,有很多种，每种有自己的识别序列（Now there are hun
13、dreds of restriction enzymes,each with its own specific recognition）sequence.,返回,概念,平头末端（blunt end）：断裂位置位于对称轴的中心；黏性末端（sticky end）：一些末端或与用同一种酶切割的其他DNA分子之间能通过碱基配对互补而退火，通常称为黏性末端。5黏性末端：即5单链延伸末端；3黏性末端：即3单链延伸末端；回文结构或回文序列（Palindrome：sequences with twofold symmetry)，是指在一个假想轴的两侧所存在的对称重复序列，即两条链的碱基顺着同一极性读（53或3
14、5），均有一样的碱基排列。,HindII recognizes this sequence:GTPyPuAC Pu:purine(A or G)CAPuPyTGPy:pyrimidines(T or C)4 bp sequence 44=256 bases6 bp sequence:more common 46=4096 bases8 bp sequence 48=65000 bases(NotI),Isocaudamer同尾酶，产生相同的粘性末端 BamHI GGATCC BalII AGATCTIsoschizomer同裂酶，识别相同的DNA序列 XmaI CCCGGG SmaI CCCG
15、GG,ExamplesAble was I ere I saw Elba(Napoleons lament)Straw?No,too stupid a fad;I put soot on warts,(a wart remedy)Go hang a salami!Im a lasagna hog.(a statement of preference in Italian food),回文结构：Palindrome(sequences with twofold symmetry),EcoRI,Pst I,回文序列的例子：,blunt end 平头末端sticky end粘性末端,计算题,从下面的
16、一段DNA上有下图示中显示的三种限制酶位点，根据限制图谱中分析：a.Hpa切割后可获得的片段数目和大小；b.EcoRI切割后可获得片段数目和大小；c.这个片段总碱基数是多少？分子量为多少Da？长度为多少nm?,Hpa,EcoRI,EcoRI,BamHI,BamHI,0.4kb,1.2kb,0.5kb,0.9kb,A piece of DNA(a plasmid or a phage DNA)that serves as a carrier in gene cloning experiments.Vectors fall into two major classes:,2.1.2.Vector
17、载体,Plasmidsphages.,载体DNA通常具备3个特性(p658)：1.含有一个复制起点，能独立于宿主染色体进行自主复制；2.含有选择标记；3.含有一个或多个限制酶的单个酶切位点；,Plasmids as Vectors,The plasmid pBR322,Plasmid（质粒）:一个宿主染色体以外的、独立的、小的环状DNA，并在寄主体内具有复制能力。A small,circular DNAs that are independent of the host chromosome and are capable of replicating in host cell.,a)分子量小
18、，3-10kb,是一个松弛型控制的复制子；;b)Containing only one cutting site for each of restriction enzymes.只有一个限制性核酸内切梅；c)Easy to be detected（易发现）.,Representative example 1（代表性的例子1）（代表性的例子2）pBR plasmid series（pBR系列）pBR322 contains genes that confer resistance to two antibiotics,ampicillin and tetracycline.Between the
19、se two genes lies the origin of replication.The plasmid was engineered to contain only one cutting site for each of several common restriction enzymes,including EcoRI,BamHI,PstI,HindIII,and SalI.,An Ideal plasmids 一个理想的质粒,克隆外部DNA使用含有PstI 位点的f pBR322 克隆(4363 bp)（foreign DNA using the PstI site of pBR
20、322）,A Mixture is produced（一个混合产物）:Plasmid（质粒）Foreign DNA（外部DNA）Recombinant DNA(重组DNA)E.coli,The recombinant plasmid no longer confers ampicillin resistance because the foreign DNA interrupts that resistance gene.,怎样才能知道重组成功呢？,Screening bacteria with recombinant DNA by replica plating,How does one d
21、o the screening?,Cells that received no DNA,or that received insert DNA only,will not be tetracycline-resistant and will fail to grow.,Cells that have received recombinant DNAs do not grow on the ampicillin plate.,影印接种法,pUC plasmid series:pUC18,pUC19ampicillin resistance gene:for selecting bacteria
22、that have received a copy of the vector.multiple cloning sites(MCS)多克隆位点（多接头）,lying within a DNA sequence of lacZpartial galactosidase gene，which provide a more convenient way of screening for clones with recombinant DNAs.(半乳糖苷酶的蓝/白斑筛选系统),Representative example 2,The host bacteria used with the pUC
23、vectors carry other partial galactosidase gene.In short,the two partial gene products can cooperate to form an active enzyme.,galactosidase can cleaves a indicator X-gal,colorless galactoside,which releases galactose plus an indigo dye that stains the bacterial colony blue.,One step screening proces
24、s:Clones with inserts:whitewhite in the presence of X-gal and grows on ampicillinClones without inserts:blue,Directional cloning Insertion of foreign DNA into two different restriction sites of a vector,such that the orientation of the insert can be predetermined.two different restriction enzymes(Ec
25、oRI and BamHI),半乳糖苷酶的蓝/白斑筛选系统,Phage is a temperate phage.It can follow two paths of reproduction.噬菌体是一种温和型的噬菌体，它可以通过两条途径进行生长。Lytic mode裂解生长Lysogenic mode溶原生长 一个包含噬菌体DNA的细菌叫溶源菌(lysogen)，这个插入的噬菌体DNA叫前噬菌体（prophage）.,Phages as Vectors 作为载体的噬菌体,Phage vectors,Advantage:Infect cells more efficiently（侵染细胞的效
26、率更高）；Accommodate much more foreign DNA(12-20kb)(high capacity：高容量)；use for constructing genomic libraries.（利用它可构建基因主文库）。,Figure 8.18 Genetic map of phage.(a)The map is shown in linear form.As the DNA exists in the phage particles;the cohesive ends(cos)are at the ends of the map.The genes are grouped
27、 primarily according to function.(b)The map is shown in circular form,as it exists in the host cell during a lytic infection after annealing of the cohesive ends.,Phage(48,513 bp),“sticky”ends（粘性末端）,The region in the middle of the phage DNA was taken out,but the genes needed for phage replication wa
28、s retained.The missing phage genes could then be replaced with foreign DNA.,insertion vectors插入型载体 replacement vectors取代型载体:DNA is removed and replaced with foreign DNA.Examples:Charon phages(see page 68)gt ZAP,Figure 4.8 Cloning in Charon 4.,Inserts（插入）:12-20 kbUse for constructing genomic librarie
29、s用它来构建基因组文库,Incomplete digestion with EcoRI was carried out for obtaining about 16-20 kb inserts.用EcoRI进行不完全的消化可得到16-20kb插入片段。,Human library（人类基因组）:500,000 clones,怎样检出呢？,概念,DNA文库是一群重组DNA分子，它们由同一种载体和不同的DNA插入片段组成。基因组文库用基因工程的方法，人工构建的含有某一生物基因组DNA的各种片段的克隆群。一般以改造的噬菌体DNA或黏粒作为载体。,See page 660-662,A labeled
30、nucleic acid probe一个标记的核酸探针Plaque hybridization滤膜杂交,See page 69,See page 661-663,杂交法可以用来鉴定DNA文库中的某一特定克隆,Cosmids as vectors（作为载体的黏粒）,Phages+plasmids=cosmids（噬菌体+质粒=黏粒）cos sites+origin of replication（黏端位点+复制原点）大容量（high capacity）40-50 kb 像噬菌体一样侵染细胞（infecting cells as a phage）像质粒一样复制（replicating as a pl
31、asmid）,Cosmids behave both as plasmids and as phages.带有黏端位点(cos)的质粒。黏粒是由人工构建的含有质粒复制子和噬菌体DNA的COS序列的载体。,M13 Phage vectors,Advantage:产生单链DNA，用于DNA测序和点突变（The vector produces single-stranded recombinant DNA,which can be used for DNA sequencing and for site-directed mutagenesis）.,噬菌体M13的DNA是单链DNA感染E.coli细
32、胞后变成双链（复制型，RF）,Phagemids(噬菌粒),The vector produces single-stranded DNA or pure RNA transcripts corresponding to either strand of a insert.,pBS,噬菌粒（phagemid）实际上是带有丝状噬菌体大间隔区的质粒载体，是集质粒和丝状噬菌体的有利特征于一身的载体，具有 ColE1 复制起点及抗生素抗性选择标记的质粒，以及丝状体噬菌体的间隔区。此间隔区含噬菌体 DNA 合成的起始与终止及噬菌体颗粒形态发生所必需的全部顺式作用序列。含噬菌粒的细菌被噬菌体感染后，基因
33、蛋白可作用于噬菌粒的间隔区，启动滚环复制产生 ssDNA 并进行包装。,最基本的根癌农杆菌的质粒：Vectors based on Ti plasmid of Agrobacterium tumefaciens 酵母人工染色体：Yeast artificial chromosomes(YACs)(see 参考1page 816-817)细菌人工染色体：Bacterial artificial chromosomes(BACs)(see参考1page 816-817)PI噬菌体人工染色体：PI phage artificial chromosomes(PACs)Expression vectors（表达载体）:A cloning vector that allows expression of a cloned gene（能够表达克隆基因的克隆载体）.Shuttle vector（穿梭载体）
