5 Stages involved in GE

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

2. Donor DNA Position of gene of interest Genetic probe 1. Isolation (a) Isolation of a specific gene from donor e.g. human • Cells broken open • Genetic probe added • Reveals position of the gene of interest

3. Bacterial cell Plasmid 1. Isolation • (b) Isolation of plasmid from a bacterial cell www.sci.sdsu.edu

4. 2. Cutting • Human DNA and plasmid DNA are cut open using the same restriction enzymes From Leaving Cert Biology

5. Recap: Restriction enzymes • DNA is cut into fragments using restriction enzymes. • One RE cuts at GAATTC • DNA from two different organisms cut with GAATTC RE, cut ends from both sources are complimentary but bind weakly to each other

6. 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 From Leaving Cert Biology

7. Recap: DNA Ligase • Gets foreign DNA to join to DNA in cloning vector • Can only work if both sources of DNA have been treated with the same restriction enzymes as cut ends will be complementary to each other • Sections of human DNA can be combined with plasmid DNA which has been cut open. • DNA Ligase forms recombinant DNA From Leaving Cert Biology

8. 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 From Leaving Cert Biology

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