BIOLOGY NOTES

CHAPTER 8: RECOMBINANT DNA TECHNOLOGY

SUBTOPIC : 8.1 Recombinant DNA Technology

LEARNING OUTCOMES :
a. Define recombinant DNA technology.
b. Define and explain the tools used in recombinant DNA technology: target DNA (gene of interest),
restriction enzymes, DNA cloning vector, host cell and modifying enzymes (DNA ligase).
c. Explain restriction enzyme and examples of enzymes that produce sticky ends (EcoRI: G↓AATTC)
and blunt ends (SmaI : CCC↓GGG).
d. Explain the characteristics of plasmid as cloning and expression vector.
e. Explain the characteristic of E.coli as host cell (bacteria) and its characteristics.
f. Explain modifying enzyme and its function:
i. DNA ligase for DNA ligation; and
ii. Taq polymerase for DNA amplification using PCR.
.
MAIN IDEAS /KEY
EXPLANATION NOTES
POINT

Formation of new combination by isolating genes from the Definition of

What is Recombinant organism and introducing them into either a similar or recombinant DNA
DNA Technology? technology
unrelated organism.

• Enable scientists to obtain copies of specific DNA

segment for the purpose of studying it.
Purpose
• Modifying the DNA of an organism to produces new
genes with new traits.

1. Target DNA (Genes of interest)

• Fragment of chromosomal to be cloned. An enzyme
must be used to cleave fragments that containing
genes of interests.
Tools Used in Cloning
• The gene of interest can be taken from chromosome of
eukaryotes or prokaryotes

1 | KMPk
2. Restriction Enzymes/ Restriction endonucleases.
• Extracted from bacteria.
• Naturally used (by bacteria) to cut viral DNA into
small fragments at specific site.
• Action: The enzyme will cut DNA at specific base
sequence called restriction sites.
• In the restriction site; most of the bases are in
palindromic sequence. Palindromic sequences?
Base sequence of one
strand reads the same as
its complement strand in
opposite direction.

A structure of cruciform hairpin DNA with a

palindromic sequence

• Most of the restriction enzyme will make staggered cut

on DNA complementary strands forming sticky end.
Example: EcoRI

EcoRI

• Some of the restriction enzyme will cut straight across

the DNA complementary strand forming blunt end.
Example: SmaI

2 | KMPk
 Restriction Enzymes Restriction Sites
5’-G↓AATTC-3’
EcoRI
3’-CTTAA↓G-5’
5’-G↓GATCC-3’
BamHI
3’-CCTAG↓G-5’
5’-GGG↓CCC-3’
SmaI
3’-CCC↓GGG-5’

SmaI

EcoRI

BamHI

3. DNA cloning vector

• A small DNA (usually isolated from prokaryote)
which a foreign DNA fragment can be inserted.
• The cloning vector carried the DNA to be cloned (gene
of interest) into the host cell.
DNA Inserts
Capacity (kb)
Types Form of vector Example
(1 kb = 1000
nucleotides)
Double
Plasmid 10 stranded pUC19
circular DNA
Bacteriophage 20 Linear DNA λ2001
Double
Cosmid 35-45 stranded sCOS-1
circular DNA

YACs – Yeast Double

Artificial 200-1500 stranded pYAC
Chromosomes circular DNA

3 | KMPk
 Characteristics of DNA cloning vector :
1. Able to accept foreign DNA in multiple cloning sites
(MCS)
2. Able to replicate freely in host cell.
Present of origin of replication initiation -ori gene
3. Possess selectable genetic marker
a. Gene that resistance to antibiotic: eg: ampR, tetR,
kanR
b. lacZ gene: encode for B- galactosidase
4. Host Cell
A cell that can be utilized from DNA cloning in order to
accept, maintain and allow the replication of cloning vector.

Characteristics:
i. Able to receive foreign DNA through the
transformation.
ii. Able to maintain the structure of DNA recombinant
from one generation to another
iii. Able to amplify the gene product from the DNA
recombinant

5. Modifying Enzymes
• Enzymes used in modification of DNA.
a) DNA Ligase
✓ Catalyzed the formation of phosphodiester bond
between adjacent nucleotides in DNA.

4 | KMPk
b) Taq polymerase
A DNA polymerase isolated (extracted) from
thermophilic bacterium Thermus aquaticus.
✓ The enzyme catalyzed the addition of free DNA
nucleotides (dNTPs) to DNA template strand
during elongation stage of PCR (DNA
amplification using PCR).
✓ Characteristic of this enzyme: able to withstand
high temperature/ heat stable
• The enzyme will not be denatured by high
temperature (during stage denaturation in
PCR).

5 | KMPk
SUBTOPIC : 8.2 Methods in Gene Cloning
LEARNING OUTCOMES :
a. Overview using diagram to show the steps in gene cloning by using plasmid.
b. Explain the steps in gene cloning by using plasmid as the vector:
i. Isolation of gene
ii. Cleave/cut
iii. Insertion
iv. Transformation and amplification; and
v. Screening (blue/white screening)
c. Explain amplification of gene involving:
i. DNA (polymerase chain reaction/PCR); and
ii. RNA (Reverse Transcription Polymerase Chain Reaction/ RT-PCR)

MAIN IDEAS
EXPLANATION NOTES
/KEY POINT

• Recombinant DNA technology can be used to insert foreign

genes into recipient cells to create recombinant plasmids.
• Recombinant plasmids can then be used to produce multiple
Introduction copies of the DNA fragment.
• 2 methods to copies (amplify) the DNA fragment:
a. Gene Cloning by using plasmid as vector
b. Polymerase Chain Reaction (PCR)
What is Gene Method for making many identical copies of gene of interest by
Cloning? inserting the gene into a living host cell (bacteria).

1. Isolation of gene
• Isolation of plasmid (as vector) from bacteria.
• Isolation of target DNA or gene of interest from sources
Eg: Human cells, Plant cells, Animal cells

Steps in Gene
Cloning by
using plasmid
as the vector
2. Cleave or cut
• Target DNA and plasmid are cut at palindromic sequence
(restriction site) with same restriction enzyme.
• For Plasmid: Plasmid opened and disrupting lacZ gene
For Target DNA: Producing thousands of fragments with
different sizes.

6 | KMPk
MAIN IDEAS
EXPLANATION NOTES
/KEY POINT

3. Insertion
• Mix DNA fragments with plasmid.
• Plasmid base pair forms hydrogen bonds with
complementary DNA fragment.
• DNA ligase is added to catalyzed the joining of DNA
fragment by forming phosphodiester bond.
• Thus, forming recombinant DNA.

• BUT, not all plasmid will join with the target DNA

Non-recombinant plasmid

7 | KMPk
MAIN IDEAS
EXPLANATION NOTES
/KEY POINT
Recombinant plasmid

4. Transformation and amplification.

• Host cells (bacteria) take up recombinant DNA after being
introduced to it.
– Bacteria and recombinant DNA are mixed in a
medium containing calcium chloride (CaCl2): CaCl2
cause the bacterial cell wall to become permeable;
bacteria can take up the recombinant DNA.
– During transformation, not all host cells will take in
the recombinant DNA; only a small proportion of
bacteria will be transformed.

8 | KMPk
 – There will be THREE conditions in the growth
medium:
✓ Colony of transformed bacteria with non-
recombinant plasmid
✓ Colony of transformed bacteria with
recombinant plasmid
✓ Colony of non-transformed bacteria

• Then, the host cells undergo amplification by reproduce

through binary fission

5. Screening
• Purpose of this step is to identify bacterial colonies that carry
gene of interest / target DNA
• Type of process: Blue-white screening
• Bacteria after transformation process will be cultured in
medium containing antibiotic (such as ampicillin) and
synthetic lactose, X-gal:
• A plasmid (vector) contains two selectable genetic markers:
1. ampR gene: encode for resistance to ampicillin and
2. lacZ gene: encode for β-galactosidase to catalyze the MCS located in lacZ gene.
hydrolysis of X-gal If gene of interest inserted
in MCS, lacZ gene will be
• The possible result of screening:
disrupted
1. Bacteria without plasmid fail to grow; because no ampR
gene, therefore not resistance to antibiotic.
2. Bacteria with plasmid live but with 2 conditions:
i. Contain non-recombinant plasmids.
• lacZ gene is functioning and hydrolyzed X-gal
producing BLUE colonies
ii. Contain recombinant plasmids (insertion of gene of
interest in MCS).
• lacZ gene is disrupted.
• X-gal NOT hydrolyzed producing WHITE colonies.

9 | KMPk
Amplification 1. Amplification of DNA fragment using PCR
of gene 2. Amplification of mRNA using RT-PCR

• A method widely used to rapidly make millions to billions of

copies complete or partial of a specific DNA sample.
• Using PCR, copies of very small amounts of DNA sequences
are exponentially amplified in a series of cycles which involve
extreme temperature changes.
Polymerase
• PCR done in thermal cycler which repeatedly change
Chain Reaction temperature of incubation according to a pre-set program.
(PCR)
• PCR components
i. DNA template (DNA to be cloned)
ii. Taq polymerase
iii. 2 strands of short DNA primer (oligo-dT)
iv. dNTPs (building blocks/nucleotides from which the DNA
polymerase synthesized a new DNA strand).

10 | KMPk
 PCR is a three-step process that is carried out in repeated cycles:
1. Denaturation (94OC):
Heat briefly to separate DNA
into single strand by breaking
hydrogen bond between the
double strand DNA. 94°C
2. Annealing (50-60OC):
Short, single stranded primer
bind to complementary
50-
Steps in PCR sequences in the single stranded 60°C
DNA.
3. Extension (72OC):
Taq polymerase adds dNTPs
according to base-pair
complementary to the 3’end
of each primer.

Reverse • RT-PCR begins by turning sample sets of mRNAs into double-

Transcription stranded DNA with the corresponding sequences.
Polymerase
• In the RT- PCR, the reverse transcriptase constructs cDNA from
Chain Reaction the mRNA in a cyclic temperature PCR reaction.
(RT-PCR)

11 | KMPk
 • The reverse transcription PCR is a good method for HIV, HPV
and Covid-19 detection. It can also detect other viral infections
too.

i. The synthesis of cDNA : reverse transcript mRNA into cDNA

• the reverse transcriptase enzyme catalysed the synthesis of first
DNA strand using mRNA as a template and short oligo-dT as a
DNA primer
• mRNA (template) then degraded by another enzyme.
• DNA polymerase catalysed the synthesizes of second DNA
strand from the 5′ to 3′ direction (using a primer in the reaction
mixture).
Steps in RT-
PCR • As result, cDNA which carries the complete coding sequence
of gene (but no intron) is produced
ii. Next in RT-PCR is the PCR step.

12 | KMPk
 BIOLOGY DIAGRAM
Steps in Gene Cloning by using plasmid as the vector

13 | KMPk
SUBTOPIC : 8.3 Application of Recombinant DNA Technology
LEARNING OUTCOMES :
a. Briefly explain applications of Recombinant DNA Technology in mass production of insulin using cDNA.
b. Describe the steps in production of insulin using cDNA.
c. State other application of DNA based biotechnology: medical, forensic, environment clean-up and
agriculture.

MAIN IDEAS
EXPLANATION NOTES
/KEY POINT

Many fields benefit from DNA technology and genetic engineering.

- Agriculture
- Forensic
- Medicine – insulin for diabetics
- Environment

Application Of
Recombinant
DNA
Technology

14 | KMPk
 ❑ cDNA is DNA molecule made in vitro using mRNA as a template
and the enzyme reverse transcriptase.
❑ A cDNA molecule lacks introns compared to DNA of the genome
❑ Can be used directly to express the proteins.
❑ cDNA is often used to clone eukaryotic genes in prokaryotes
❑ cDNA is used because:
❑ cDNA represents the expression of the genes without the
introns.
Complementary
❑ there is no process for RNA splicing in bacteria cell.
DNA (cDNA)

1. mRNA for insulin is isolated from the beta cells of islets of

Langerhans in the pancreas.
2. Reverse transcriptase enzyme is added to synthesis a cDNA by using
mRNA as template. mRNA strand then discarded by using mRNA-
degrading enzyme.
3. DNA polymerase enzymes is added and synthesizes a second DNA
strand, complementary to cDNA in vitro.
4. Restriction enzyme (BamHI ) is used to cut the cDNA and isolate

Production of just the sequence that encodes for the insulin protein.
insulin 5. Plasmid removed from a bacterial cell is cut at a specific site using
the same restriction enzyme (BamHI)
6. The cDNA is inserted to the plasmid using DNA ligase. The
recombinant plasmid carrying the human DNA for insulin.
7. The recombinant DNA then transform into the host (E. coli) by
transformation.
8. The bacterium E. coli with its recombinant plasmids, is allowed
to reproduce and undergo screening process.

15 | KMPk
 9. Finally, a lot of bacterial clones carrying many copies of the gene
for insulin will be produced.

Steps in production of insulin using

cDNA

mRNA for insulin is isolated Reverse transcriptase DNA polymerase enzymes is

from the ß-cells of islets of
Langerhans in the pancreas.
enzyme is added to
synthesis a cDNA by using
mRNA as template. mRNA
strand then discarded by
using mRNA-degrading
enzyme.
added and synthesizes a
second DNA strand,
complementary to cDNA in
vitro.

Plasmid removed from a bacterial cell Restriction enzyme (BamHI) is

is cut at a specific site using the same used to cut the cDNA and
restriction enzyme (BamHI) isolate just the sequence that
encodes for the insulin protein.

The cDNA is inserted to the

The recombinant DNA then
plasmid using DNA ligase. The
transform into the host (E. coli)
recombinant plasmid carrying
by transformation.
the human DNA for insulin.

Gene expression
Finally, a lot of bacterial clone The bacterium E. coli with its
occurs in E. coli and
carrying many copies of the recombinant plasmids, is
produced insulin
gene for insulin will be allowed to reproduce and
produced. undergo screening process.

Insulin is separated and 16 | KMPk

purified to produce
human insulin
17 | KMPk