Explain the t
recomb
1QUESTION
tools used in DNA
binant technology
[10 marks]
1
TARGET DNA/GENE OF
INTEREST
Sequence of DNA contain
is taken from any source s
animal.
Cell containing gene
of interest
Gene of
interest
ning gene of interest that
such human, plant and
2
RESTRICTION ENZYME
Enzyme that is isolated fro
recognizes the specific ba
DNA at restriction site by b
phosphodiester bond and
palindromic.
om bacteria to
ase sequence and cut the
breaking the
the base sequence are
Fig. 20-3-1 Restri
DNA 5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.
Enzyme EcoR1 from bacteria E
sequence 5' GAATTC 3' and
cut 5' GAATTC 3'
Enzyme BamH1 from bacteria _
sequence 5' ______ 3' and
cut 5' _______ 3'
iction site
3
5
Sticky end
Escherichia coli recognize base
__________ recognize base
3
DNA CLONING VECTOR
DNA molecule used in gen
foreign DNA fragment (ge
genome of a host cell and
Plasmid
R
Bacterium
ne cloning to carry
ene of interest) into a
d replicate there.
3
DNA CLONING VECTOR
e.g
Plasmids are small cir
molecules that replicate
the bacterial chromoso
Plasmid
R
Bacterium
rcular DNA
e separately from
ome
4
HOST CELL
Organism/cell that receiv
DNA for cloning purpose
transformation process
ve recombinant
e through
5
MODIFYING ENZYME
Enzyme that join DNA fragment
interest and plasmid vector formin
form the phosphodiester bon
backbones. Example: DNA ligase
permanently between gene of
ng recombinant DNA/plasmid by
nd between sugar-phosphate
Fig. 20-3-3 Restri
DNA 5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.
2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One poss
3 DNA ligase
seals strands.
Recombin
iction site
3
5
Sticky end
e
sible combination
nant DNA molecule
Explain the tools used in DNA recomb
The first tool involve in DNA recombinant tec
is a sequence of DNA containing gene of interest that
animal.
The restriction enzyme, the enzyme that is is
sequence and cut the DNA at restriction site by breaki
are palindromic.
The DNA cloning vector is molecule used in g
interest) into a genome of a host cell and replicate the
The other tools involve is the host cell, that is
cloning purpose.
The last tools involve is modifying enzyme. T
gene of interest and plasmid vector forming recombina
phosphodiester bond between sugar-phosphate backb
binant technology [10 marks]
chnology is target DNA or gene of interest. This DNA
is taken from any source such human, plant and
solated from bacteria to recognizes the specific base
ing the phosphodiester bond and the base sequence
gene cloning to carry foreign DNA fragment (gene of
ere.
s organism or cell that receive recombinant DNA for
This enzyme join DNA fragment permanently between
ant DNA or recombinant plasmid by form the
bones.
Characteristics of host cell
1. Can accept recombinant plasmid
or recombinant DNA through
transformation process
2. Able to maintain the structure of
recombinant plasmid/DNA (from
generation to generation)
3. Able to amplify/express the gene
from recombinant plasmid/DNA
4. Antibiotic sensitive/resistance
5. Can clone itself or recombinant
plasmid
21 Recombinant
DNA (plasmid)
Bacterium/host cell Plasmid put into
bacterial cell through
transformation process
Host cell grown in culture
3 to form a clone of cells
containing the “cloned”
gene of interest
Protein expressed
by gene of interest
2
Rewrite all t
paragraph f
one compl
the point in
form to get
lete essay
Discuss the d
plasmid and
3QUESTION
differences between
d cosmid as cloning
vector
[8 marks]
Points
Plasmid is a circular DNA molecule whi
replicate independently from the host
chromosome while cosmid is a hybrid
between a plasmid and a phage lambda
chromosome.
Plasmid is a single type of cloning vecto
while cosmid is a hybrid cloning vector
Plasmid can accommodate up to 15kb
cosmid can accommodate in a range of
50 kb
Example of plasmid is pUC18 while exa
of cosmid is sCOS-1
ich can
a
or
while
f 35 –
ample
Describe th
4QUESTION
he steps involve in
gene cloning.
[10 marks]
Fig. 20-4-1
TECHNIQUE
1 Bacterial cell
lacZ gene
Restriction
site
ampR gene Bacterial
plasmid
ISOLATION OF GENE
1. Isolate DNA that contain gene
donor cell
2. isolate bacterial plasmid as a
Hummingbird
cell
Sticky Gene of interest
ends
Hummingbird
DNA fragments
e of interest from eukaryotic
a vector from bacterial cell
Fig. 20-4-1
TECHNIQUE
2 Bacterial cell
lacZ gene
Restriction
site
ampR gene Bacterial
plasmid
CLEAVE/CUT DNA DONO
Both target DNA and plasmid are
compatible restriction enzyme.
Produce sticky/blunt ends which
ensure successful combination.
Cut plasmid at lacZ gene so lacZ
Cut target DNA to get gene of in
Hummingbird
cell
Sticky Gene of interest
ends
Hummingbird
DNA fragments
OR AND PLASMID
e cleaved by using the same and
h complementary each other to
Z gene is disrupted.
nterest.
Fig. 20-4-2
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene Bacterial
plasmid
3
Recombinant
INSERTION OF DNA FRAG
The gene of interest inserted int
Both gene of interest and plasm
ligase by catalysing the formatio
The product is now called recom
DNA
Hummingbird
cell
Sticky Gene of interest
ends
Hummingbird
DNA fragments
Nonrecombinant
plasmid
t plasmids
GMENT INTO PLASMID
to the plasmid
mid are joined by using DNA
on of phosphodiester linkage
mbinant plasmid/ recombinant
Fig. 20-2a
Bacterium
1 Gene in
plasmid
Bacterial Plasmid
chromosome
Recombinant
DNA (plasmid)
Recombinant
bacterium
Cell containing gene
of interest
nserted into
d
Gene of DNA of
interest chromosome
2
2 Plasmid put into
bacterial cell
Fig. 20-3-1 Restri
DNA 5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.