Transcript PCR - جامعة الملك سعود

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