5 stages involved in ge 1.isolation 2.cutting 3.ligation and insertion 4.transformation 5.cloning...
TRANSCRIPT
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