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DrM4ScienceGrin
ds1. Isolation
2. Cutting
3. Ligation and Insertion
4. Transformation
5. Cloning & Expression
DrM4ScienceGrin
ds
Donor DNA
Genetic probe
(a) Isolation of a specific gene from donor e.g. human
• Cells broken open
• Genetic probe added
• Reveals position of the gene of interest
Position of gene of interest
DrM4ScienceGrin
ds (b) Isolation of plasmid from a bacterial cell
Bacterial cell
Plasmid
www.sci.sdsu.edu
DrM4ScienceGrin
ds Human DNA and plasmid DNA are cut open using the same restriction enzymes
From
Leaving Cert B
iology
DrM4ScienceGrin
ds 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
DrM4ScienceGrin
ds 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 B
iology
DrM4ScienceGrin
ds 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 B
iology
DrM4ScienceGrin
ds 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 B
iology
DrM4ScienceGrin
ds 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