Isolation

ds
ir n
1.

2. Cutting
eG
3.
n c
Ligation and Insertion

c ie
Transformation
S
4.

M 4 Cloning & Expression

r
5.

D
 (a) Isolation of a specific gene from donor e.g. human

ds
• Cells broken open
ir n
• Genetic probe added

eG
•
n c
Reveals position of the gene of interest

c ie
4 S Genetic probe

Position of
gene of
rM
interest

D Donor DNA
 s
(b) Isolation of plasmid from a bacterial cell


ir n d
Bacterial

eG cell

n c
c ie Plasmid

4 S
rM
D www.sci.sdsu.edu
 Human DNA and
ds
ir n
plasmid DNA are
cut open using the
same restriction
G

From Leaving Cert Biology

enzymes

c e
ie n
S c
M 4
Dr
 DNA is cut into
s


fragments using
restriction enzymes.
ir n d
 One RE cuts at GAATTC

eG
c
 DNA from two different
organisms cut with

ie
GAATTC RE, cut endsn
S c
from both sources are
complimentary but bind

4
weakly to each other

M
Dr
 The target gene is placed in the
ds
ir n
DNA of the plasmid/cloning
vector and joins on to it

eG
When cut plasmids are mixed
c


n
with cut human DNA, different

ie

From Leaving Cert Biology

combinations result.

S c


M 4
DNA ligase is used to form strong
bonds within the recombinant
DNA
Dr
 Gets foreign DNA to join to DNA in
s

cloning vector
 Can only work if both sources of
ir n d
G
DNA have been treated with the

e
same restriction enzymes as cut

c
ends will be complementary to each

From Leaving Cert Biology

other

c ie
 Sections of human DNA can be

S
combined with plasmid DNA which

M 4
has been cut open.
 DNA Ligase forms recombinant
DNA

Dr
 Uptake of recombinant DNA into cell
s


ir n d

From Leaving Cert Biology

eG
n c
c ie
4 S
M
Vast majority of cells Some cells Some cells

r
Special techniques will identify the small
Dnumber of bacteria with the target gene
Cloning: Identical copies of the bacteria with the target gene are produced

ds
ir n
eG
n c
c ie
4 S
rM
D
Expression: Getting the organism with the recombinant
DNA to produce the desired protein