5 Stages involved in GE 1 Isolation 2

5 Stages involved in GE 1. Isolation 2. Cutting 4 M 4. r D 5. G e c n e i c S 3. ir n Ligation and Insertion Transformation Cloning & Expression s d

1. Isolation (a) Isolation of a specific gene from donor e. g. human ir n • Cells broken open • Genetic probe added • Reveals position of the gene of interest c n G e 4 M r D Position of gene of interest e i c S s d Genetic probe Donor DNA

1. Isolation (b) Isolation of plasmid from a bacterial cell ir n Bacterial cell c n G e e i c S r D 4 M www. sci. sdsu. edu Plasmid s d

2. Cutting r D 4 M e i c S c n G e ir n s d From Leaving Cert Biology Human DNA and plasmid DNA are cut open using the same restriction enzymes

Recap: Restriction enzymes DNA is cut into fragments using restriction enzymes. 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 One G e c n r D 4 M e i c S ir n s d

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 r D 4 M e i c S From Leaving Cert Biology c n G e ir n s d

Recap: DNA Ligase Gets c n G e r D 4 M e i c S ir n s d From Leaving Cert Biology 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

4. Transformation Uptake of recombinant DNA into cell c n e i c S 4 M techniques will identify the small Special r Dnumber of bacteria with the target gene Vast majority of cells Some cells From Leaving Cert Biology G e ir n s d

5. Cloning & Gene Expression Cloning: Identical copies of the bacteria with the target gene are produced c n G e r D 4 M e i c S ir n s d Expression: Getting the organism with the recombinant DNA to produce the desired protein