5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression

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5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression Slide 2 Donor DNA Genetic probe (a) Isolation of a specific gene from donor e.g. human 1. Isolation Cells broken open Genetic probe added Reveals position of the gene of interest Position of gene of interest Slide 3 1. Isolation (b) Isolation of plasmid from a bacterial cell Bacterial cell Plasmid www.sci.sdsu.edu Slide 4 2. Cutting Human DNA and plasmid DNA are cut open using the same restriction enzymes Slide 5 3. Ligation The target gene is placed in the DNA of the plasmid/cloning vector and joins on to it When cut plasmids are mixed with cut human DNA, different combinations result. DNA ligase is used to form strong bonds within the recombinant DNA Slide 6 2. Cutting & 3. Ligation Slide 7 4. Transformation Uptake of recombinant DNA into cell Vast majority of cells Some cells Some cells Special techniques will identify the small number of bacteria with the target gene Slide 8 4. Transformation Slide 9 5. Cloning & Gene Expression Cloning: Identical copies of the bacteria with the target gene are produced Expression: Getting the organism with the recombinant DNA to produce the desired protein