5 Stages involved in GE

1. Isolation

2. Cutting

3. Ligation and Insertion

4. Transformation

5. Cloning & Expression

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

1. Isolation• (b) Isolation of plasmid from a bacterial cell

Bacterial cell

Plasmid

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2. Cutting• Human DNA and

plasmid DNA are cut open using the same restriction enzymes

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

2. Cutting & 3. Ligation

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

4. Transformation

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