Recombinant DNA technology

and PCRBy

Said AbbadiProfessor 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 disorders3. Cancer detection4. 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 - Requirements for Recombinant DNARecombinant DNA Technique: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.

Gene Cloning

1- Insert

3- Cloning vector e.g. Plasmid

2- Restriction endonuclease

4- DNA Ligase

5- Host cell

Host Chromosome

Cloning Vectors

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

- Types of cloning vectors:1. Plasmids:2. Bacteriophages3. 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