基因克隆與亞克隆.ppt

1、專題：基因克隆與亞克隆,DNA cloning makes molecular biology possible,Molecular biology seeks to explain the relationships between the structure and function of biological molecules (DNA, RNA, protein, other small regulators) and how these relationships contribute to the operation and control of biochemical proc

2、esses (replication, transcription, translation),Molecular analysis of proteins, RNA and DNA, and/or their interaction is essential for molecular biology,Getting enough large biomolecules for molecular analysis is impossible by direct purification or isolation from the cell directly,DNA cloning make

3、it possible,DNA cloning is 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, forming RECOMBINANT DNA, which can be replicates independently of the original genome, and normally

4、 in other host species. 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.,What is DNA cloning?,Prepare interested DNA fragment from a genome (insert),Plasmid preparation (vector),Restriction

5、digestion (trimming the DNA ends),Ligation (join the insert and the vector),Transformation & selection (introduce the plasmids into host cells),Assay of the recombinant DNA: (1) is it a right clone? G4 (2) Analyze the biological function of the cloned gene/DNA,DNA Cloning: a simplified flow chart,G2

6、,G3,G4,G4,H,I,PCR or Genomic DNA,To insert a fragment of DNA into a vector is a relatively simple process: Restriction enzyme linearize the plasmid. A restriction enzyme cleaved a target DNA to generate potential insert DNAs,vector DNA has been cut with the same enzyme. DNA ligase to link the compat

7、ible ends of the two DNAs.,1973 - Boyer, Cohen & Chang,Transform E. coli with recombinant plasmid,Stanley Cohen & Annie Chan,Herbert Boyer,Kanamycin resistance gene,Plasmid pSC101,Tetracycline resistance gene,E. coli transformed with recombinant plasmid,Transformed cells plated onto medium with kana

8、mycin and tetracycline,Only cells with recombinant plasmid survive to produce colonies,Vector,Target DNA,Recombinant plasmid/DNA,A simple example of DNA Cloning,Clone,Transfer of a fragment of cloned DNA from one vector to another. Enables us to investigate a short region of a large cloned fragment

9、in more detail. To transfer a gene from one plasmid to a vector designed to express it in a particular species.,Subcloning,Prepare the interested DNA fragment from cloned DNA (insert),Plasmid preparation (vector),Restriction digestion (trimming the DNA ends),Ligation (join the insert and the vector)

10、,Transformation & selection (introduce the plasmids into host cells),Assay of the recombinant DNA,DNA subcloning: a flow chart,PCR or Plasmid, phage,Ligation, transformation and analysis of recombinants,Alkaline phophatse DNA ligation & recombinant DNA molecules Transformation & selection Transforma

11、tion efficiency 4.Screening transformants 5.Growth and storage of transformants Gel analysis Fragment orientation,Alkaline phophatse removes the phosphate groups from th 5-ends of the vector DNA linearized by a single restriction enzyme to prevent the self-ligation of the vector DNA upon the followe

12、d ligation,DNA ligation Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5-ends have phosphate groups.,X if the vector is phosphoralated,Recombinant DNA molecules,The use of alkaline phosphate to prevent religation of vector molecules,Transformation and se

13、lection,Competent cells: E. coli cells treated with Ca2+ solution are susceptible to take up exogenous DNA. Enzymes involved in host cell defending, such as restriction-modification system are suppressed. Transformation: a process of uptake of exogenous DNA by competent cells. Heat-shock: After the

14、DNA is uptaken, the cells shall be put at 42oC for 1 min in order to induce the suppressed enzymes for cell defending,Growth and storage of transformants,Can be grow in liquid broth or solid plates. Maintain the selection pressure by the presence of the corresponding antibiotics. Store the transform

15、ant bacteria by freezing a portion in the presence of glycerol to protect from ice crystal formation.,Gel analysis and fragment orientation,Distinguish the recombinant plasmids from the recreated vectors by Size of the plasmids Restriction digestion 2.Determine the orientation of a inserted DNA frag

16、ment cloned by a single enzyme by restriction digestion that cuts asymmetrically within the insert sequence, and once at a specific site of the vector,Selection with antibiotic resistanc (ampr),Transformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 103 to

17、more than 108. 105 is adequate for a simple cloning.,Hosts,Host organism/cell: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning. Prokaryotic host: E. coli (most cases) Eukaryotic host: Yeast (Saccharomyces cerevisiae), mammalian, insect,Inserts,DNA cloning: PCR amplification of the interest