LECTURE NOTE WEEK 13

CHAPTER 8:
RECOMBINANT DNA TECHNOLOGY

SUBTOPIC

1. Recombinant DNA
Technology

2. Methods in
Gene Cloning

8.3 Application of Recombinant DNA
Technology

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.

Definition of DNA Technology

Formation of new combinations of genes
by isolating genes from the organism

and introducing them into either a similar
or unrelated organism.

Enable scientists to obtain many Purpose of
copies of specific DNA segment Recombinant
for the purpose of studying it
DNA
Technology

Modifying the DNA of an
organism to produce new genes

with new trait

This technology has multidisciplinary applications and potential to deal with
important aspects of life; improving health, enhancing food resources, and

resistance to divergent adverse environmental effects.

TOOLS USED IN RECOMBINANT DNA
TECHNOLOGY

TARGET DNA/ RESTRICTION DNA CLONING HOST CELL MODIFYING
GENE OF ENZYME
INTEREST ENZYME VECTOR

TARGET DNA (GENE OF INTEREST)

A selected DNA that contain gene of
interest.

Can be obtain from cell of various
organism; example: bacterial cell,
plant cell, animal stem cell

RESTRICTION ENZYME

A molecular scissors that cut the single
strand or the double strand DNA at specific
point.

Extracted from bacteria; naturally used to
cut viral DNA into small fragments at
specific base/site (for defense mechanism).

Restriction Enzymes (RE) Source
EcoRI Escherichia coli
BamHI Bacillus amyloliquefaciens
SmaI Serratia marcescens

RESTRICTION ENZYME Enzymes Restriction Sites
EcoRI
The enzyme splice through DNA at BamHI 5’-GAATTC-3’
specific base sequence called as SmaI 3’-CTTAAG-5’
restriction sites.
5’-GGATCC-3’
Bases in restriction site are 3’-CCTAGG-5’
palindromic.
5’-GGGCCC-3’
A palindromic sequence in DNA is 3’-CCCGGG-5’
sequence of base pair that is identical
on both strand if read in 5’ to 3’
direction.

EcoR1: make staggered cut in two Sma1: cut straight across both
strands forming sticky end strands forming blunt ends

DNA CLONING VECTOR

A cloning vector is a small piece of DNA
that can be stably maintained in an
organism, and into which a foreign DNA
fragment can be inserted for cloning
purposes.

The cloning vector is a DNA taken from a
virus, the cell of a higher organism, or it
may be the plasmid of a bacterium

DNA CLONING VECTOR

A large number of cloning vectors are
available, and choosing the vector may
depend upon a number of factors, such
as the size of the insert (DNA).

Types DNA Inserts Form of vector Example
Capacity
Plasmid Double stranded pUC18
Bacteriophage (1 kb = 1000 circular DNA λ2001
Cosmid nucleotides) Linear DNA sCOS-1
YACs – Yeast
Artificial 10 kb Double stranded pYAC
Chromosomes circular DNA
20 kb
35-45 kb Double stranded
circular DNA
200-1500 kb

DNA CLONING VECTOR: PLASMID

• Small ring-shape DNA in
bacteria.

• Not part of chromosome.
• Self-replicating.
• Has small number of

genes.
• Example: pUC18

CHARACTERISTICS OF PLASMID AS CLONING
AND EXPRESSION VECTOR

1. Able to accept foreign DNA in multiple
cloning sites (MCS).

2. Able to replicate freely in host cell.
Present of Ori gene

3. Possess selectable genetic marker.
 Importance for screening purpose.

a) Antibiotic resistance gene
Example: ampR, tetR,kanR

b) lacZ gene

DNA CLONING VECTOR: BACTERIOPHAGE

• Viruses that infect bacteria
• Can carry larger DNA

inserts than bacterial
plasmid
• Eg. λ2001

DNA CLONING VECTOR: COSMID

• Hybrids of plasmid and
bacteriophage
lambda DNA.

• Can accommodate larger
inserts of DNA

• example: Scos-1

DNA CLONING VECTOR: YEAST ARTIFICIAL
CHROMOSOMES

• Plasmid with yeast
chromosome

• Example: pYAC –
capacity is bigger
than other vectors

HOST CELL

A cell that can be
utilized for DNA cloning
in order to accept,
maintain and allow the
reproduction of cloning
vector.

Example: bacteria

CHARACTERISTIC OF E. coli (BACTERIA)
AS HOST CELL

1. Able to receive DNA 3. Able to amplify the
recombinant through gene product from the
the transformation DNA recombinant.
process.

2. Able to maintain the
structure of DNA
recombinant from one
generation to other.

MODIFYING ENZYME (DNA LIGASE FOR DNA LIGATION)

Enzymes used in modification of DNA by
catalyzed the formation of phosphodiester
bonds between adjacent nucleotides of DNA
(gene of interest and plasmid) .

Produced recombinant DNA/ recombinant
plasmid

MODIFYING ENZYME (Taq POLYMERASE FOR DNA
AMPLIFICATION USING PCR)

Taq polymerase is a thermostable DNA
polymerase named after the thermophilic
eubacterial Thermus aquaticus.

It is frequently used in the polymerase chain
reaction (PCR), a method for greatly
amplifying the quantity of short segments of
DNA