walk in Biology

1QUESTION

Explain the tools used in DNA
recombinant technology

[10 marks]

1 Cell containing gene
of interest

TARGET DNA/GENE OF Gene of
INTEREST interest

Sequence of DNA containing gene of interest that
is taken from any source such human, plant and
animal

2

RESTRICTION ENZYME

Enzyme that is isolated from bacteria to
recognizes the specific base sequence and cut the
DNA at restriction site by breaking the
phosphodiester bond and the base sequence are
palindromic.

Fig. 20-3-1 Restriction site

DNA 5 3
3 5

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end

Enzyme EcoR1 from bacteria Escherichia coli recognize base
sequence 5' GAATTC 3' and
cut 5' GAATTC 3'

Enzyme BamH1 from bacteria __________ recognize base

sequence 5' ______ 3' and

cut 5' _______ 3'

3 Plasmid

DNA CLONING VECTOR Bacterium

DNA molecule used in gene cloning to carry
foreign DNA fragment (gene of interest) into a
genome of a host cell and replicate there.

3

Plasmid

DNA CLONING VECTOR Bacterium

e.g
Plasmids are small circular DNA
molecules that replicate separately from
the bacterial chromosome

4

HOST CELL

Organism/cell that receive recombinant
DNA for cloning purpose

5

MODIFYING ENZYME

Enzyme that join DNA fragment permanently between gene of
interest and plasmid vector forming recombinant DNA/plasmid by
form the phosphodiester bond between sugar-phosphate
backbones. Example: DNA ligase

Fig. 20-3-3 Restriction site

DNA 5 3
3 5

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end

2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.

One possible combination
3 DNA ligase

seals strands.

Recombinant DNA molecule

Explain the tools used in DNA recombinant technology [10 marks]

The first tool involve in DNA recombinant technology is target DNA or gene of interest. This DNA
is a sequence of DNA containing gene of interest that is taken from any source such human, plant and
animal.

The restriction enzyme, the enzyme that is isolated from bacteria to recognizes the specific base
sequence and cut the DNA at restriction site by breaking the phosphodiester bond and the base sequence
are palindromic.

The DNA cloning vector is molecule used in gene cloning to carry foreign DNA fragment (gene of
interest) into a genome of a host cell and replicate there.

The other tools involve is the host cell, that is organism or cell that receive recombinant DNA for
cloning purpose.

The last tools involve is modifying enzyme. This enzyme join DNA fragment permanently between
gene of interest and plasmid vector forming recombinant DNA or recombinant plasmid by form the
phosphodiester bond between sugar-phosphate backbones.

2QUESTION

Define cloning vector and host
cell. Describe the characteristics

of cloning vectors and host cell

[10 marks]

DNA CLONING VECTOR

Bacterium Cell containing gene
of interest

1 Gene inserted into
plasmid

Bacterial Plasmid

chromosome Gene of
interest
Recombinant DNA of
DNA (plasmid) 2 chromosome

2 Plasmid put into
bacterial cell

Recombinant
bacterium

DNA molecule used in gene cloning to carry foreign DNA
fragment (gene of interest) into a genome of a host cell
and replicate there.

Characteristics of vector Plasmid

1. Able to accept foreign gene/DNA at Bacterium
specific restriction site/multiple cloning
sites/MCS 4 3 1
2
2. Able to be cloned/replicated Bacterium/host cell
independently inside host cell because
plasmid contain Ori (origin of replication
initiation)

3. Has antibiotic of specific gene/selectable
genetic marker that codes for antibiotic
resistance e.g ampicillin resistance gene
(ampR)

4. Able to carry foreign DNA into
bacteria/host cell

HOST CELL

Organism/cell that receive recombinant
DNA for cloning purpose

21 Recombinant
DNA (plasmid)

Characteristics of host cell Bacterium/host cell Plasmid put into
bacterial cell through
1. Can accept recombinant plasmid transformation process
or recombinant DNA through
transformation process Host cell grown in culture

2. Able to maintain the structure of 3 to form a clone of cells
recombinant plasmid/DNA (from containing the “cloned”
generation to generation) gene of interest

3. Able to amplify/express the gene Protein expressed
from recombinant plasmid/DNA by gene of interest

4. Antibiotic sensitive/resistance 2

5. Can clone itself or recombinant
plasmid

Rewrite all the point in
paragraph form to get
one complete essay

3QUESTION

Discuss the differences between
plasmid and cosmid as cloning
vector

[8 marks]

Points

Plasmid is a circular DNA molecule which can
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 vector
while cosmid is a hybrid cloning vector

Plasmid can accommodate up to 15kb while
cosmid can accommodate in a range of 35 –
50 kb
Example of plasmid is pUC18 while example
of cosmid is sCOS-1

4QUESTION

Describe the steps involve in
gene cloning.

[10 marks]

Fig. 20-4-1

TECHNIQUE Hummingbird
cell

1 Bacterial cell
lacZ gene

Restriction Sticky Gene of interest
site ends
Hummingbird
ampR gene Bacterial DNA fragments
plasmid

ISOLATION OF GENE

1. Isolate DNA that contain gene of interest
2. isolate bacterial plasmid as a vector from E. coli

Fig. 20-4-1

TECHNIQUE Hummingbird
cell

2 Bacterial cell
lacZ gene

Restriction Sticky Gene of interest
site ends
Hummingbird
ampR gene Bacterial DNA fragments
plasmid

CLEAVE/CUT DNA DONOR AND PLASMID

Both target DNA and plasmid are cleaved by using the
same and compatible restriction enzyme.
Cut plasmid at lacZ gene so lacZ gene is disrupted.
Cut target DNA to get gene of interest.

Fig. 20-4-2

TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest
site ends
Hummingbird
ampR gene Bacterial DNA fragments
plasmid
3
Nonrecombinant
plasmid

Recombinant plasmids

INSERTION OF DNA FRAGMENT INTO PLASMID

The gene of interest inserted into the plasmid

Both gene of interest and plasmid are joined by using DNA
ligase

The product is now called recombinant plasmid/
recombinant DNA

Fig. 20-2a

Bacterium Cell containing gene
of interest

1 Gene inserted into
plasmid

Bacterial Plasmid

chromosome Gene of
interest
Recombinant DNA of
DNA (plasmid) 2 chromosome

2 Plasmid put into
bacterial cell

Recombinant
bacterium

Fig. 20-3-1 Restriction site

DNA 5 3
3 5

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end

Fig. 20-3-2 Restriction site

DNA 5 3
3 5

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end

2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.

One possible combination

Fig. 20-3-3 Restriction site

DNA ligase DNA 5 3
is an 3 5
enzyme that
seals the 1 Restriction enzyme
bonds cuts sugar-phosphate
between backbones.
restriction
fragments Sticky end

2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.

One possible combination
3 DNA ligase

seals strands.

Recombinant DNA molecule

Fig. 20-4-3

TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest
site ends
Hummingbird
ampR gene Bacterial DNA fragments
plasmid
4
Nonrecombinant
plasmid

Recombinant plasmids

Bacteria carrying
plasmids

TRANSFORMATION AND AMPLIFICATION

Fig. 20-2a

Bacterium Cell containing gene
of interest

1 Gene inserted into
plasmid

Bacterial Plasmid

chromosome Gene of

Recombinant interest DNA of
DNA (plasmid)
2 chromosome

2 Plasmid put into

bacterial cell by transformation

Recombinant
bacterium

4 TRANSFORMATION AND AMPLIFICATION

The recombinant plasmids are then
inserted back into bacterial cells (as host)
by transformation process.

Followed by amplification process where
the recombinant plasmid is amplified in
the host cell
Produce genetically identical copies of
recombinant plasmid

5 BLUE-WHITE SCREENING

Host bacteria are cultured on nutrient
medium containing ampicilin and sugar
called X-gal

Ampicilin is used to identify the bacterial
cells that has taken up plasmids

X-gal is used to identify the bacterial cells
with recombinant plasmid

5 BLUE-WHITE SCREENING

BLUE WHITE

• Bacterial cells carry non- • Bacterial cells carry
recombinant plasmid recombinant plasmid

• lacZ gene is intact and • lacZ gene is disrupted
can encoded β- and cannot encoded β-
galactosidase enzyme galactosidase enzyme
and hydrolyse X-gal and cannot hydrolyse X-
sugar gal sugar

Fig. 20-4-4

TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest
site ends
Hummingbird
ampR gene Bacterial DNA fragments
plasmid

Nonrecombinant
plasmid

Recombinant plasmids

Bacteria carrying
plasmids

RESULTS Colony carrying recombinant
plasmid with disrupted lacZ gene
Colony carrying non-
recombinant plasmid One of many
with intact lacZ gene bacterial
clones

Screening for Clones Carrying a Gene of
Interest

• A clone carrying the gene of interest can be
identified with a nucleic acid probe having a
sequence complementary to the gene

• This process is called nucleic acid
hybridization

Fig. 20-7 Probe Gene of
DNA interest
TECHNIQUE
Single-stranded Film
Radioactively DNA from cell
labeled probe

molecules

Multiwell plates
holding library
clones

•

Nylon membrane

Location of Nylon
DNA with the membrane

complementary

sequence

5QUESTION

Explain the Polymerase Chain

Reaction (PCR)

[10 marks]

8.2 : (ii) Polymerase Chain Reaction

8.2 : (ii) Polymerase Chain Reaction

1. PCR is a technique to amplify specific
DNA sequence

2. Through in vitro process
3. PCR involves 3 steps cycle

4. First step is denaturation / separation od double
stranded DNA
5. By heating at high temperature to about 95oC

6. Second step is annealing of primer
7. Temperature is lowered to about 50oC

8. To allow the primers to form hydrogen bond with
complementary bases at DNA template

9. Third step is extension / elongation /
polymerization
10. Temperature is increased at 72oC
11. Complementary DNA strand is synthesized in 5’
to 3’ direction by the addition of free DNA nucleotide

12. Catalysed by Taq polymerase

13. Taq polymerase is heat stable / resistant

14. This cycle is repeated 30-40 times

6QUESTION

Discuss the process of cloning
a human gene for insulin
production in bacteria.

[10 marks]

Fig. 20-6-1

DNA in
nucleus

mRNAs in
cytoplasm

1. mRNA that codes for insulin is extracted from
human pancreatic cells.

2. mRNA is used as template in transcription

Fig. 20-6-2

DNA in
nucleus
mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA

DNA Primer
strand

3. mRNA is incubated in the test tube with reverse
transcriptase enzyme

Fig. 20-6-3

DNA in
nucleus

mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA

Degraded DNA Primer
mRNA strand

4. By using reverse transcriptase enzyme, first cDNA
strand is produced.

5. Followed by enzymatic degradation/hydrolysis of
mRNA

Fig. 20-6-4

DNA in
nucleus

mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA

Degraded DNA Primer
mRNA strand

DNA
polymerase

6. Second cDNA strand complementary to the first is synthesized ,
catalyzed by DNA polymerase

Fig. 20-6-5 DNA in
nucleus
7. Formation of double
strand cDNA mRNAs in
8. The cDNA carries cytoplasm
complete sequence
(exon) of the gene Reverse
without introns. transcriptase Poly-A tail
mRNA

Degraded DNA Primer
mRNA strand

DNA
polymerase

cDNA

Fig. 20-2a

Bacterium

1 Gene inserted into
plasmid

Bacterial Plasmid

chromosome Gene of
interest
Recombinant cDNA
DNA (plasmid) 2

2 Plasmid put into
bacterial cell

Recombinant
bacterium

9. Ligation of adaptor DNA carrying a restriction site.
10. Isolate plasmid (vector) from bacteria E. coli.
11. Cut both plasmid DNA and cDNA with the same restriction
enzyme

Fig. 20-2a

Bacterium

1 Gene inserted into
plasmid

Bacterial Plasmid

chromosome Gene of
interest
Recombinant cDNA
DNA (plasmid) 2

2 Plasmid put into
bacterial cell

Recombinant
bacterium

12. cDNA fragment is inserted into the plasmid and ligation by using
DNA ligase producing the recombinant DNA plasmid

Fig. 20-2a

Bacterium

1 Gene inserted into
plasmid

Bacterial Plasmid

chromosome Gene of
interest
Recombinant cDNA
DNA (plasmid) 2

2 Plasmid put into
bacterial cell

Recombinant
bacterium

13. transformation of recombinant DNA plasmid into host cell (E.coli )

Recombinant
bacterium

Gene of 3 Host cell grown in culture
Interest to form a clone of cells
Copies of gene containing the “cloned”
gene of interest
Basic
research Protein expressed
on gene by gene of interest

Protein harvested

4 Basic research and Basic
various applications research
on protein

14. Screening of recombinant colonies is performed
15. Amplification is performed whereby induced the expression of the clone
genes.
16. Insulin is extracted from E.coli