《分子生物学》2-section g gene manipulation-1.ppt
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1、DNA Cloning DNA cloning: DNA克隆 Molecular cloning: 分子克隆 Gene manipulation: 基因操作 Recombinant DNA technology: 重组DNA技术 Genetic engineering 遗传工程,基因工程 Biotechnology: 生物技术 (Gene engineering, Enzyme engineering, Cell / Fermentation engineering, Biomedicine, Agrobiotechnolgy etc.),DNA cloning (Gene manipulat
2、ion) To place a relatively short fragment of a genome, which might contain the gene or other sequence of interest, in an autonomously replicating piece of DNA, known as a vector (载体), forming recombinant DNA, which can be replicated independently of the original genome, and normally in other host sp
3、ecies altogether. Propagation of the host organism containing the recombinant DNA forms a set of genetically identical organism, or a clone. This process is called DNA cloning.,Basic procedure of DNA cloning,Vector,Genomic fragment (restriction, PCR), cDNA (insert),Plasmid preparation (vector),Restr
4、iction digestion (trimming the DNA ends),Ligation (join the insert and the vector),Transformation (introduce the plasmids into host cells),Analysis of the recombinants,Electrophoresis (check your DNA),DNA Cloning: a simplified flow chart,Isolation and manipulation of fragments of an organisms genome
5、,Molecular analysis of proteins or other interested gene products,Impossible by direct purification,DNA cloning,Impossible by direct isolation,Crucial !,Make all possible !,Gene manipulation, molecular cloning, genetic engineering,Applications of DNA cloning,Sequencing, hence to derive protein seque
6、nce; genomics Isolation and analysis of gene promoter etc Investigation of protein/enzyme/RNA function in various forms. Identification mutations, genetic diseases Biotechnology: proteins of pharmaceutical importance Transgenic plants and animals Gene therapy,DNA Cloning, Techniques & Applications,S
7、ection G: Gene manipulation (DNA cloning & subcloning, & basic techniques) section H: Cloning vectors Section I: Gene libraries & screening Section J: Analysis & uses of cloned DNA,The most basic concepts and technical tools of molecular biology,G1 DNA cloning: an overview (basic concepts) G2 Prepar
8、ation of plasmid DNA G3 Ligation, transformation and analysis of recombinants,Section G Gene manipulation,G1 DNA cloning: an overview,Hosts and vectors Subcloning DNA libraries Screening libraries Analysis of a clone,back,Plasmid as vectors,Plasmids (质粒): small, extrachromosomal circular molecules,
9、from 2 to 200 kb in size, which exist in multiple copies within the host cells. contain an origin of replication and replicate independently Usually carry a few genes, one of which may confer resistance to antibacterial substance. Example: ampR gene encoding the enzyme b-lactamase which degrades pen
10、icillin antibiotics such as ampicillin; kanR for kanamycin.,back,Earlier plasmid developed,Versatile cloning plasmid,Phagemid (噬菌粒),Hosts and vectors,Host organism/cell: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning. Hosts for DNA cloning vector Prokar
11、yotic host : E. coli ( most cases) Eukaryotic host : Yeast Saccharomyces cerevisiae (large fragments of human genome),General features of a Vector autonomously replicating DNA independent of hosts genome. Easily to be isolated from the host cell Most are circular, some are linear Contains at least o
12、ne selective marker, which allows host cells containing the vector to be selected amongst those which do not. Contains a multiple cloning site (MCS),Types of vectors,Cloning vectors Expression vectors Integration vectors Viral vectors,Cloning vectors: allowing the exogenous DNA to be inserted, store
13、d, and manipulated at DNA level. E. coli cloning vector: plasmids, bacteriophages (l and M13), plasmid-bacteriophage l hybrids (cosmids). Yeast cloning vector: yeast artificial chromosomes (YACs),Expression vectors: allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for
14、 RNA transcription are required. bacterial expression vectors yeast expression vectors mammalian expression vectors,Integration vectors: allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation. The integration is either random insertion or conducted by
15、homologous recombination between the homologous sequence shared by the plasmid and the genome of the recipient cells. bacterial integration vectors (Agrobacterium tumefaciens Ti plasmid is used to integrate DNA into plant genome) yeast integration vectors Mammalian integration vector: gene targeting
16、,back,Viral vectors:. Bacterial phage: Lambda, M13 Insect: baculoviruses Mammalian viruses: SV40, pox virus, adenovirus, retroviruses Plant viruses: TMV, PVX,back,Adenoviral vector system,Plant virus vector: PVX (potato virus X),Subcloning Transfer of a fragment of cloned DNA from one vector to anot
17、her. Enables us to investigate a short region of a large cloned fragment in more detail. To transfer a gene from one plasmid to a vector designed to express it in a particular species.,Preparation of plasmids containing a cloned DNA fragment (insert),Plasmid preparation (vector),Restriction digestio
18、n (trimming the DNA ends),Separation, purification, ligation (join the insert and the vector),Transformation & selection of transformants (introduce the plasmids into host cells),Analysis of the recombinants,DNA Subcloning: a flow chart,Restriction endonuclease,back,Agrose Gel Electrophoresis: check
19、 your DNA at each step Separation and Purification of DNA fragments of interests Analysis of recombinant plasmids,ladder,Restriction analysis of a plasmid,DNA libraries,DNA libraries are sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector follo
20、wed by propagation in the host. A clone is a genetically distinct individual or set of identical individuals Genomic libraries cDNA libraries,Genomic libraries prepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNA,c
21、DNA libraries DNA copies (cDNA) synthesized from the mRNA by reverse transcription are inserted into a vector to form a cDNA library. Much more efficient in identifying a gene, but do not contain DNA coding for functional RNA or noncoding sequence.,Screening libraries,Colony or plaque hybridization:
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