PCR

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PCR

• Presented by
• Rana AL-Turki

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

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Definition of PCR

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

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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.

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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.

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Requirements for PCR

• 3- Heat stable polymerase.
• 4-Four d NTPs.
• 5- Buffer(10x).
• 6-Thermocycler
• Change temperature very rapidly for each cycle.

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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.

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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.

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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.

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