Presented by : Mostafa al-mosavi

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Presentation transcript:

Presented by : Mostafa al-mosavi PCR Presented by : Mostafa al-mosavi

1993 Nobel Prize in Chemistry

1- Definition of PCR. 2- Requirements for PCR. 3-PCR Process. 4-Procedure.

Definition of PCR Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence in two hours.

Requirements for PCR 1- DNA sample : Very small amount (ng or some times less). 2- Two primers: (Forward)a. b. (Reverse) You must know the sequence of the flaking regions so you can order appropriate primers.

Thermus aquaticus 3- Heat stable polymerase. The bacterium Thermus aquaticus was first discovered in several springs in the Great Fountain area of the Lower Geyser Basin at Yellowstone National Park

4-Four dNTPs.(Deoxy nuleoside tri phosphates) Nitrogen Base dNTPs Adenine Thymine Guanine Cytosine

Requirements for PCR 5- Buffer(10x).mgcl2 6- DDW

Hot Star Tag Polymerase Procedure Volume(µl) Reagents 2.5 10x PCR buffer 2 dNTPs 0.6 Forward Primer Reverse Primer 0.3 Hot Star Tag Polymerase 17 Distilled water DNA template 25 Total volume

7-Thermocycler: Change temperature very rapidly for each cycle.

The cycling reactions There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

PCR Process PCR –reaction is divided into 3 steps: 1-Denatration: During denatration, the template DNA is seprated into its 2 separate by heating at the temperature about 95ºC.

PCR Process 2-Anneling: This involves the annealing of the primer to the denatured. 3-Extension: The synthesizing ,take place at a temperature of around 72ºC,this corresponds to the optimal temperature for the Tag-polymerase to work.