NOTA KULIAH RECOMBINANT TECHNOLOGY

BIOLOGY UNIT BIOLOGY SB015
KOLEJ MATRIKULASI MELAKA SEMESTER 1
SESSION 2020/2021

CHAPTER 8:

RECOMBINANT DNA TECHNOLOGY

8.1 RECOMBINANT DNA TECHNOLOGY
8.2 METHODS IN GENE CLONING

8.3 APPLICATION OF RECOMBINANT DNA
TECHNOLOGY

JPU PSPM SEMESTER I:

Paper 1 Paper 2

7 marks 20 marks

CHAPTER 8 : BIOLOGY SB015
RECOMBINANT DNA TECHNOLOGY SEMESTER 1
SESSION 2020/2021

8.1 RECOMBINANT DNA TECHNOLOGY

LEARNING OUTCOMES

(a) Define recombinant DNA technology.
(b) Define and explain the tools used in recombinant

DNA technology.
(c) Explain restriction enzyme and examples of

enzymes that produce sticky ends and blunt
ends.
(d) Explain the characteristic 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.

RECOMBINANT DNA TECHNOLOGY
DEFINITION:

The techniques involved in altering the
characteristics of an organism by inserting
genes from another organism into its DNA.

Resistance to herbicides

RECOMBINANT DNA TECHNOLOGY

• Also known as

genetic engineering

• Usually produce by

gene cloning

• This altered DNA
• known as

recombinant
DNA

RECOMBINANT DNA
DEFINITION:

DNA which contain foreign gene
⮚ Foreign gene is originated from different species

Foreign gene

Recombinant DNA

TOOLS USED IN RECOMBINANT DNA
TECHNOLOGY

1) Target DNA (gene of interest)
2) Restriction enzyme
3) DNA Cloning vector
4) Host cell

5) Modifying enzyme (DNA ligase)

TOOLS USED IN RECOMBINANT DNA
TECHNOLOGY

1. Target DNA 3. DNA
containing gene cloning
vector
of interest

4. Modifying 2. Restriction
enzyme enzyme

(E.g. DNA ligase) 4. Host
cell

1. TARGET DNA / TARGET GENE

DEFINITION:
A fragment of DNA containing gene of

interest to be cloned

Cell containing
gene of interest

Gene of
interest DNA of

chromosome
(“foreign” DNA)

2. RESTRICTION ENZYME

DEFINITION:
A type of enzyme that can cleave molecules

of DNA at restriction site

• Also known as restriction endonuclease
• Produce by many bacteria

2. RESTRICTION ENZYME

10

RESTRICTION SITE

Restriction site contain palindromic
sequences

PALINDROMIC SEQUENCE

• Is the same sequence of bases which can

be
read from both opposite directions on

double
stranded DNA

• As the recognition site for restriction
enzyme

WHAT DO YOU NOTICE ABOUT THESE
PHRASES?

i. radar
ii. racecar
iii. Madam I’m Adam
iv. a man, a plan, a canal, Panama
v. Was it a bar or a bat I saw?

2. RESTRICTION ENZYME

Function :
- Recognise and cut / cleave DNA at restriction

site //
- Breaks phosphodiester bond
- To produces sticky ends or blunt ends

✎ cut out foreign DNA into
fragments at restriction

site

✎ cut open bacterial plasmid
at restriction site

2. RESTRICTION ENZYME

HOW TO CUT the DNA strands?

1) In a staggered way, creating

sticky ends

2) In a straight way, creating
blunt ends

Hanging
complementary
single strands

DNA

Example of Restriction Enzymes
that produce sticky ends:

Enzymes Producer Restriction sequence
(organism)

1. EcoRI E. coli RY 13 ✂

5’– G A A T T C – 3’
3’– C T T A A G – 5’

✂

Example of Restriction Enzymes
that produce blunt ends:

Enzymes Producer Restriction sequence
(organism)

✂

1. SmaI Serrana 5’– C C C G G G – 3’
marcescens 3’– G G G C C C – 5’

✂

5’– C C C G G G – 3’
3’– G G G C C C – 5’

Blunt ends

The importance of restriction enzyme on
bacteria

1.Degraded viral / foreign DNA
2.Defend against virus
3.Prevent replication of DNA viral

What would happen if the restriction
enzyme in bacterial cells fail to function

i) STICKY ENDS

• A double-stranded
fragment has at least
one single stranded
end or staggered cut

• The unpaired bases
will form hydrogen
bond with compatible
sticky ends of other
DNA molecules;
created by same
restriction enzyme.

ii) BLUNT ENDS

• A fragment of DNA in which there are
no unpaired bases or overhangs in the end

• Both strand are of the same length

3. CLONING VECTOR

DEFINITION:
An agent / small DNA molecule that carry

gene of interest into host cell in
genetic engineering

Cloning vector
e.g. plasmid

3. CLONING VECTOR

• Have two or more restriction sites @ multiple

cloning sites (MCS)

• Have origin of replication (ori)
• Have a selection marker genes for screening

e.g. ampR

HOW CAN CLONING VECTOR HELP US?

• A way to get genes into
bacteria easily
– Insert target gene into
plasmid (vector)
– Forming recombinant
plasmid
– Transfer recombinant
plasmid into bacteria
(host cell)
– Bacteria now expresses
new gene
– bacteria make new protein

TYPES OF CLONING VECTOR

Cloning vector

Plasmid Cosmid

Bacteriophage / Yeast artificial
phage chromosome (YAC)

PLASMID

e.g. pUC18

✔ Small, circular (ring-shaped) bacterial DNA
✔ Not a part of bacterial chromosome
✔ Able to replicate freely
✔ Can carry 10-15 kilobases of base pairs of

foreign DNA

BACTERIOPHAGE / PHAGE

e.g. λ2001

✔ Virus that infect bacteria
✔ by injecting its genetic material into bacteria
✔ Also known as phage
✔ Can carry 20 kilobases of base pairs of

foreign DNA

COSMID

e.g. sCOS-
1

✔ bacterial plasmid which is inserted with

‘cos’ gene

✔ ‘cos’ gene is originated from λ phage
✔ can carry up to 44 kilobases of base pairs of

foreign DNA

‘cos’
gene

Plasmid Cosmid

YEAST ARTIFICIAL CHROMOSOME(YAC)

e.g. pYAC

✔ Unicellular fungi
✔ Easy to grow and have plasmid
✔ Can carry more than 100 kilobases and up to

1000 kilobases of base pairs of foreign DNA

CHARACTERISTICS OF CLONING VECTOR

1. Able to accept foreign DNA in MCS
- Contain two or more restriction sites /
Multiple cloning site (MCS)

WHAT IS MULTIPLE CLONING SITES???
✔ Short segment of DNA
✔ containing many restriction sites
✔ where many restriction enzymes can cut

✔ and many foreign DNA fragments can be

inserted

PSPM 05/06 , 07/08

MULTIPLE CLONING SITE (MCS)

Multiple
Cloning
Sites (MCS)

CHARACTERISTICS OF CLONING VECTOR

2. Able to replicate freely / independently in host
cell
- contain origin of replication (ori)

PSPM 05/06 , 07/08

CHARACTERISTICS OF CLONING VECTOR

3. Possess selectable genetic marker
(i) ampR gene (ampicillin resistance gene)
- Resist to antibiotic
(ii) lacZ gene
- Encode for β-galactosidase

- enable cells containing recombinant DNA
to be identified during screening @ easy to
be selected

PSPM 05/06 , 07/08

CHARACTERISTICS OF CLONING VECTOR

4. Able to express or amplify the cloned gene
under suitable conditions

PSPM 05/06 , 07/08

4. HOST CELL
DEFINITION:
Cell that receive the recombinant DNA for
cloning purposes

e.g. Genetically engineered E.coli

Host cell

CHARACTERISTICS OF HOST CELL

1. Able to accept / receive the recombinant DNA
through transformation.
WHAT IS TRANSFORMATION???
A process by which recombinant DNA is
transferred into host cell.

PSPM 05/06

CHARACTERISTICS OF HOST CELL

2. Able to maintain the recombinant DNA from one

generation to another

3. Able to amplify the gene product from the
recombinant DNA

✔able to produce

multiple copies of
recombinant DNA
(when bacteria
reproduce through
binary fission)

4. Able to express gene of interest within

recombinant DNA

5. MODIFYING ENZYME

DEFINITION:
Enzymes involved in genetic engineering to
degradation, synthesis and alteration of the

nucleic acids

MODIFYING ENZYME : DNA Ligase

• Join the foreign DNA fragments containing

gene of interest and bacterial plasmid

• by catalyzing the formation of

phosphodiester bond

• forming recombinant DNA

LIGATION INTO PLASMID

phosphodiester bond

MODIFYING ENZYME : Taq polymerase

•Thermostable/ heat-stable polymerase
• used in Polymerase Chain Reaction (PCR)
• replaced the DNA polymerase because of the

temperature conditions of PCR
• purified from hot spring bacteria:

Thermus aquaticus

Function:
- to add DNA nucleotides to the 3’ end of

primer in order to produce new DNA strand

REFERENCE

Campbell, N. A. & Reece, J.B., Urry, L.A., Cain
M.L., Wassermen, S.A., Minorsky, P.V. &
Jackson, R. B. 2015. Biology. (10th Edition).
Pearson Benjamin-Cummings. USA. (page
450-452)