5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning &...
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Transcript of 5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning &...
![Page 1: 5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression.](https://reader036.fdocuments.us/reader036/viewer/2022062620/551af13b5503462e578b49b6/html5/thumbnails/1.jpg)
5 Stages involved in GE
1. Isolation
2. Cutting
3. Ligation and Insertion
4. Transformation
5. Cloning & Expression
![Page 2: 5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression.](https://reader036.fdocuments.us/reader036/viewer/2022062620/551af13b5503462e578b49b6/html5/thumbnails/2.jpg)
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
![Page 3: 5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression.](https://reader036.fdocuments.us/reader036/viewer/2022062620/551af13b5503462e578b49b6/html5/thumbnails/3.jpg)
1. Isolation• (b) Isolation of plasmid from a bacterial cell
Bacterial cell
Plasmid
www.sci.sdsu.edu
![Page 4: 5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression.](https://reader036.fdocuments.us/reader036/viewer/2022062620/551af13b5503462e578b49b6/html5/thumbnails/4.jpg)
2. Cutting• Human DNA and
plasmid DNA are cut open using the same restriction enzymes
![Page 5: 5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression.](https://reader036.fdocuments.us/reader036/viewer/2022062620/551af13b5503462e578b49b6/html5/thumbnails/5.jpg)
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
![Page 7: 5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression.](https://reader036.fdocuments.us/reader036/viewer/2022062620/551af13b5503462e578b49b6/html5/thumbnails/7.jpg)
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
![Page 9: 5 Stages involved in GE 1.Isolation 2.Cutting 3.Ligation and Insertion 4.Transformation 5.Cloning & Expression.](https://reader036.fdocuments.us/reader036/viewer/2022062620/551af13b5503462e578b49b6/html5/thumbnails/9.jpg)
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