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recombinant DNA technology_1920

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Published by sallehattun.salleh, 2019-09-18 07:04:56

Recombinant DNA Walk in

recombinant DNA technology_1920

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.






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