Recombinant

DNA technology
and PCR
By

Said Abbadi
Professor of medical microbiology & Immunology

OUTCOMES
Define recombinant DNA and recombinant DNA technology

Name the applications of recombinant DNA technology

Define PCR
Enlist the basic steps of doing PCR

Provide examples of the uses of PCR

Polymerase chain reaction

Target amplification (The PCR
reaction) :
Used to amplify something
found in such small amounts
that without PCR it would be
undetectable.

Polymerase chain reaction ( PCR)

PCR is a prime mediated, temperature
dependent
technique
for
enzymatic
amplification of a specific sequence (target
sequence) to such an extent that it can be
detected.
The technique can be used to detect very small
amounts of specific nucleic acid material in
clinical specimens where bacterial, viral, or
fungal agents are thoughts to play a causative
role.

Polymerase chain reaction

It is based on repeated cycles of
A. High temperature template denaturation
B. Oligonucleotide prime annealing
C. polymerase mediated extension

FOUR ingredients are needed:

1. Target DNA (to be amplified)
2. Primer
3. Polymerase enzyme
4. Nucleotides

PCR steps
1.

Heat at 94oC (Denaturation)

2.

Annealing by reducing temperature to 55oC,

promoting primers to attach themselves to
either ends of the target strip.

3.

Primer extension: polymerase enzyme triggers

the formation of new DNA from nucleotides.
Extension is done by taq polymerase.

4.

When the temperature is raised again, the new

strands separate and the process begin again

 Steps in PCR reaction (step 1)

Denaturation
separate parent strands in preparation to the new
strand synthesis

 Steps in PCR reaction (step 2)

Annealing:
stick primers to the parent strands to prime DNA
synthesis
DNA synthesis requires a primer to start DNA synthesis

 Steps in PCR reaction (step 3)

Extension

addition of nucleotides, one at a time, to the

growing end of the DNA strand (3 end) using the
parent strand as the template

cycle through 25-35 times

Applications of PCR
1. Diagnosis of infections due to:

bacteria, viruses, fungi and protozoa.

2. Diagnosis of inherited disorders
3. Cancer detection
4. In medico legal cases.

GENETIC RECOMBINATION
(GENE CLONING)

GENETIC RECOMBINATION
This is a recent technology (also called
recombinant DNA tech. ,Genetic engineering or
gene cloning) done in vitro where isolated DNA
segments from different species like bacteria
,virus, animal cell are joined together to form
new combinations.
Definition:
The ability to join genes (DNA fragments ) coding
for certain protein from different species.

- Requirements for Recombinant

DNA Technique:
1- DNA of interest (Insert).
2- A cloning vector.
3- Restriction endonuclease enzymes.
4- DNA ligase.
5- A biological host cell

Steps of gene cloning

1-DNA is extracted ,cleaved by RE (insert).
2-Vector cleaved by the same RE.
3-The insert is ligated to vector( plasmid) by
ligase enzyme. (recombinant plasmid).
4- The recombinant plasmid is introduced
into a host cell (by transformation) in
which it will replicate.

3- Cloning
vector e.g.
Plasmid

1- Insert
2- Restriction
endonuclease
4- DNA Ligase

Host
Chromosome

Gene Cloning

5- Host cell

Cloning Vectors
These are vehicles used to carry and introduce foreign
DNA fragments into a host cell.

- Types of cloning vectors:

1. Plasmids:
2. Bacteriophages
3. Cosmids
4. Animal viruses
Ideal cloning vector must be :
1- As small as possible
2-Well characterized for at least one RE.
5-carry a selectable marker (Antibiotic resistance gene)
(gene location and nucleotide sequence)
3-Capable of autonomous replication within host cell
4-Possess a single cleavage site

The best cloning vectors are:

1. Plasmids: These are very useful vectors as they fulfill
most of the above characters plus the fact that they can
easily transfer from one cell to another.
2. Bacteriophages: The DNA of the lambda phage is
used to carry foreign genes into E. coli by transduction
3. Cosmids These are circular double stranded DNA
molecues which are artificially constructed from
plasmid DNA and phage DNA to be used as cloning
vectors. Cosmids can efficiently carry large genes that can
not be carried by either plasmid or phage alone.
Animal viruses: adenovirus, pox, vaccinia, HSV

Restriction Endonucleases
- Definition:
These are enzymes that recognize and restrict (cleave)
specific nucleotide sequences within DNA molecules at
both strands of DNA at internal positions within these
sequences.

- Source:
They are obtained from a wide variety of bacteria and
a few are obtained from fungi and algae.

- They are named according to their source e.g. EcoRl

enzyme is derived from E. coli and recognizes the
sequence GAATTC.

Host Organisms
- Microorganisms commonly used as hosts for

cloning experiments include some bacteria

and fungi.
- Strains used for this purpose should be:
1- Free of any restriction enzyme activity
that might degrade foreign DNA.

2- The nucleic acid of the host be easily

separable from that of the cloning vector by
physical or chemical means (high advantage) .
3- High metabolic rate and high replication
rate.

Applications of Recombinant DNA

Technology
I- Diagnostic applications:
a- Genetic researches and gene mapping of
microorganisms.
b- Preparation of Nucleic acid (N.A.) probes.
II- Therapeutic applications:
1- Production of biological products of medical
importance, e.g. hormones, interferons,
interleukins, monoclonal antibodies ... etc.
2- Production of recombinant vaccines, e.g.
hepatitis B vaccine.
3- Gene therapy.

Thank you
Dr Azza Abdulazim