中南大学 生物化学 考研课件 ch13 重组DNA 克隆和嵌入基因的形成 recombinant DNA06.ppt

1、Chapter 13,Recombinant DNA: Cloning and Creation of Chimeric Genes 重组DNA: 克隆和嵌入基因的形成,Content内容,Basic knowledge 基础知识 Cloning vectors 克隆载体 Genomic DNA library / cDNA library (基因组DNA文库/cDNA文库) Expression vectors (表达载体) PCR (聚合酶链式反应) Application of recombinant DNA technology (重组DNA技术的应用),Some common ter

2、ms一些常见术语,DNA/Gene cloning 基因克隆 Vector /Plasmids 载体/质粒 Gene engineering 基因工程 Gene manipulation 基因操纵 Recombinant DNA 重组DNA Transgene/Transgenic animal(plant) 转基因/转基因动(植)物 cDNA/Genomic DNA library cDNA/基因组(总)DNA文库,self-replication 自我复制 host cell/E. coli 宿主细胞 /大肠杆菌 virus / l phage 病毒/l噬菌体 foreign DNA 外源

3、DNA recombinant DNA重组DNA cohesive ends 粘性末端 reanneal 重退火 antibiotic resistance gene 抗抗菌素基因 reverse transcription 反转录 reverse transcriptase 反转录酶 encoded protein 编码蛋白质 oligonucleotide寡核苷酸,General procedure Of DNA cloning (DNA克隆的基本过程),由外源DNA与载体形成重组DNA,重组DNA转入宿主细胞复制,筛选带重组DNA的细菌细胞,培养筛选的细菌细胞,分离复制的重组DNA,从重

4、组DNA中分离外源DNA,Some enzymes used in recombinant DNA Technology 重组DNA技术中常用的工具酶,Vector 载体,Vector An agent that can carry a DNA fragment into a host cell 载体是能承载DNA 并能将其转入某宿主细胞的物质 Cloning vector 克隆载体 The vector that is used for reproducing the DNA fragment 用于复制DNA片段的载体 Expression vector 表达载体 The vector th

5、at is used for expressing certain gene in the DNA fragment 用于表达某DNA片段中基因的载体 Commonly used vectors 常用载体 plasmid, l phage, cosmid and yeast artificial chromosome (YAC) 质粒, l噬菌体,装配型质粒酵母人造染色体,The essential conditions for an agent to be a cloning vectors 成为克隆载体的条件,A cloning site 克隆位点 A polylinker which c

6、an recognize several different restriction enzymes 具有能识别一些不同限制性(内切)酶的多连接头(酶切位点) A selectable marker 选择性标识 At least an antibiotics-resistance gene for selective amplification 具有至少一个抗抗菌素基因用于复制子的选择 A replicator 复制子 A replication origin (ORI) for proliferation in the host cell 具有一个能在宿主细胞中进行复制的复制起始子,A ty

7、pical plasmid vector典型的质粒载体,(多限制性酶切位点),复制起始子,抗氨苄西林基因片段,1977年构建成功的pBR322,pBR322,粘性末端,也叫cohesive end,平滑末端,在克隆载体中导入更多的酶切位点,Directional cloning 定向克隆,P.401,The typical bacterial transformation experiment 典型的细菌转化实验,See P.402,噬菌体作为克隆载体,BAC 载体 Bacterial Artificial Chromosomes,YAC 载体 Yeast Artificial Chromos

8、omes,DNA cloning 流程图,利用pBR322在大肠杆菌中克隆外源DNA,DNA libraries (DNA文库),Definition Collection of cloned DNA fragments. Each clone in the library is called a genomic DNA clone (克隆出的DNA片段的集合，文库中的每一个克隆叫做一个基因组DNA克隆) Role useful for searching a DNA of interest 用于寻找目的（感兴趣的）DNA Types Genomic DNA libraries 基因组DNA文

9、库 a genomic DNA library is made from the genomic DNA of an organism cDNA libraries 互补DNA文库 collection of complementary DNA libraries,Number of clones needed 所需克隆数,P: probability几率,一般取0.99 N: number of clones 克隆数 f: faction containing a particular fragment (含有特征片段的分数),N=ln(1-P)/ln(1-f ),f=克隆的片段长度/总DN

10、A长度,Screening DNA libraries （筛选DNA文库）,The way to screen DNA library By hybridization with a DNA probe (that is complementary to part of the nucleotide sequence of the desired gene). DNA 探针 Review on DNA probe A DNA restriction fragment (DNA限制性片段) Cloned DNA （克隆DNA） A synthetic oligonucleotide （合成寡核苷

11、酸）,Screening the genomic DNA library,For the case of using a plasmid vector, screening by Colony lift （菌落转移） Use agar plates to bear (culture) bacterial clones Overlay each agar plate with a nitrocellulose membrane to form a replica of the plate in that some of the colonies will have adhered to it a

12、nd in the same pattern as the colonies on the plate The following procedure: Lyse the bacterial cells and denature the DNA by alkali Hybridize the denatured DNA with a radiolabeled DNA probe Wash away unreacted probe Take Autoradiography of the filter For the case of using a bacteriophage vector, sc

13、reening by Plaque lift (嗜菌斑转移) Technique and procedure are the same as that for colony lift,Labeled DNA probe,Screening a genomic library by colony hybridization (or plague hybridization) 通过菌落杂交(或嗜菌斑杂交)筛选基因组文库,菌落(或嗜菌斑)模板,Replicate onto Nitrocellulose disc,Treat with NaOH neutralize, dry,Autoradiogra

14、ph film,Darkening Identifies clonies (plaques) containing DNA desired,Design of DNA probe from sequence of amino acids 从氨基酸序列设计DNA探针,Screening the cDNA library,Method Similarly by hybridization. 用类似的杂交法 Alternatively method 另外的方法 using a ligand of the expressed protein, e.g., antibody that binds to

15、the target protein 采用表达蛋白的配体,如与目标蛋白键合的抗体 Note in this case, the labeled antibody called expression vector, and the library made from which is called expression cDNA library 在这种情况下,标识抗体叫表达载体,由此组成的文库叫表达cDNA文库.,cDNA文库能否构建成功的关键是高质量的mRNA的获得 一个好的cDNA文库的特征 包含有足够大量的独立克隆，代表最初的mRNA样品中的所有转录本; 克隆中包含接近全长的mRNA.,I

16、solation of eukaryotic mRNA via olig(dT)-cellulose chromatography 通过寡聚(dT)-纤维素色谱分离真核信使RNA,从真核mRNA合成cDNA,表达载体应有的元件,大肠杆菌表达载体所需元件,A typical expression-cloning vector 典型表达-克隆载体,A typical expression vector for the synthesis of a hybrid protein 用于合成一个杂合蛋白的典型表达载体,Reporter Gene 报告基因,Two reporter proteins,b-

17、galactosidase (半乳糖苷酶) Expressed under inducing of Ptac by galactose analog, IPTG (iso-propyl-b-thio-galactoside异丙基-b-硫代半乳糖苷) Hydrolyzes X-gal and form a product of blue color Green fluorescent protein(绿色荧光蛋白),Blue,b-galactosidase,Polymerase chain reaction (PCR) 聚合酶链式反应,Word and terms: Flanking seque

18、nce: 侧翼序列 Thermophilic bacteria: 嗜热菌 Taq polymerase: Taq 聚合酶 （Taq: Thermus aquaticus） Primer:引物 Oligonucleotide: 寡聚核苷酸 Denature: 变性 Anneal： 退火 Elongation: 延伸 Founder Mullis K, a scientist in PE-Cetus corporation, USA, invented PCR at 1985,Principles of PCR,Conditions required for PCR,Nucleic acid te

19、mplate: a target double-stranded DNA (length: 100-300bp optimal for detection. It may be up to 10-20kb if Taq polymerase is used; Concentration: 102-105copies; 1mg genomic DNA or 1ng E. Coli DNA 3*105 copies) Oligonucleotide, i.e., two primers that hybridize to flanking sequences on opposing strands

20、 of the target (final conc. in PCR: 0.2-1mmol/L. It is in large excess over parental DNA) dNTPs, i.e., all four deoxyribonucleoside triphosphates (final conc. in PCR: 20-200mmol/L) A DNA polymerase, e.g., Taq polymerase Suitable buffer (most often 10-50mmol/L Tris-HCl, pH 8.3-8.8, 50mmol/L KCl) Mg2+

21、(0.5-5.0mmol/L) Recycling parameters (recycling times: normally 30 cycles; temperature and time for each denaturation, annealing and elongation see below.),Application of PCR (PCR的应用),Amplifying a single DNA molecule from a complex mixture 从混合物中扩增某一DNA分子 Sequencing DNA Inducing point mutations, deletions or insertions of target DNA (点突变,目标DNA分子的删除或插入) Amplifying vanishingly small a