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