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

2. 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
Genetic probe
The first step involves breaking open the cells of the donor to release the DNA and isolate the gene of interest e.g. insulin producing gene.
Cells are broken open using chemicals and enzymes e.g. washing up liquid
Donor DNA is extracted
Genetic probe is added
A DNA probe consists of a small fragment of DNA labelled with an enzyme, a radioactive tag or a flurescent dye tag.
The probe will bind to a complementary DNA sequence by base pairing.
Identifying the presence and location of the gene of interest
Position of gene of interest
Donor DNA

3. 1. Isolation (b) Isolation of plasmid from a bacterial cell
The DNA from the bactterial cell is released.
A bacterial cell contains a circular loop of DNA called a plasmid.
The plasmid is isolated from the bacterial cell.
The plasmid will act as a vector for carrying a new gene i.e. the gene from the donor will be inserted into the plasmid DNA..

4. 2. Cutting
Human DNA and plasmid DNA are cut open using the same restriction enzymes
From Leaving Cert Biology
The donor DNA and plasmid DNA are cut using enzymes called restriction enzymes.
Restriction enzymes recognise specific sequence of bases
Act as a molecular scissors to cut the DNA strand within the recognition sequence.
The donor DNA and plasmid DNA are cut using the same restriction enzymes.
Bacteria use restriction enzymes to defend themselves against attacks from bacterial viruses. The enzymes cut invading viral DNA and render it harmless.

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
Universality of genetic code
Plasmid will produce the polypeptide coded for by the donor DNA
Expression: Getting the organism with the recombinant DNA to produce the desired protein