PCRPCR Presented by : Rana AL-Turki. 1- Definition of PCR. 2- Requirements for PCR. 3-PCR Process. 4-Procedure.

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Presentation transcript:

PCRPCR Presented by : Rana AL-Turki

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 approximately two hours.

Principle of the PCR The purpose of a (PCR) is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing.

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

Requirements for PCR 3- Heat stable polymerase. 4-Four d NTPs. 5- Buffer(10x). 6-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.

Procedure Volume(µl)Reagents 2.510x PCR buffer 2dNTPs 0.6Forward Primer 0.6Reverse Primer 0.3Hot Star Tag Polymerase 17Distilled water 2DNA template 25Total volume