PCR TECHNIQUE

1
????? ????? ??????? ????????Polymerase Chain
Reaction (PCR)

• ?.?.?. ???? ???? ????
• ????? ????? ?????? ???????
• ???? ???????

2
????? ??? ??????? ????

• ???? ?????? ??????? ???? ????? ?????? ??? ??????
  ?????? ???? ?????? ? ???? ???? ?? ???? ???? ?????
  ??????? ???? ?????. ? ???? ???? ????? ?????????
  ??? 1000 ????? ?????????? ???????? ( ???? ??????
  ?????? ) ? ?? ??? ????? ???? ?? ?????? ?? ???
  ??????? ????????? ??? .
• ??? ?????? ?? ???? ??????????? ??????? ????? ????
  ??? ??????? ?? ????? ?????? (DNA ) ???? ????? ?
  ?????? ??? ??????? ??? ?? ?????? ?? ????? ??
  ????? ???? ??? ?????? ???? ????? ?????? (DNA )
  ???? ????? ???? ?????? ?????? (??????).

3
????? ???????
???? ?????? ????? ??? ????? ??????DNA ???? ???
????? ??? ?????? ????? ??? ?????? ??????? ??????
?.???? ???? Kary Mullis ???? ???? ????? ???? ??
???? ????? ?????? DNA ????? ??? ??? ????? ..
????? ?? ??? 1993 ? ????? ???? ?? ????????.
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?? ?? ????? ??? PCR ?

• ?? ????? ??????? ???? ??? ???? ????? ??? ????? ??
  ??? ????? ?????? DNA ?? ??????? (in vitro) ????
  ???? ???? ??????? ??????? Thermal Cycler ????
  ???? ??????? ??? 95 ???? ???? ?????? ????? ??????
  ??? ????? ....
• ??????? ??????? ????? ??? ??? ????? ??? ?????
  ???? ????? ?? ?????? ???????
• ????? ????? ????? ?????????? ? ???????
  ????????? ????????.
• ???? ??? ???? ????? ??? ??????

5
(No Transcript)
6
???? ??????? ?????
7
??????? ????? ????? ??????? ????????

• ?????? ??????? ???? ????? ???? ?? ????? ???????
  ???????? ???????? ( ???????? , ??????????
  ???????? , ????????? ... ?????? ) ???????
  ??????? ???????? .
• ????? ?? ??????? ???????? ???? ?? ???? ???
  ?????? ??? ?????? ?????? ?????
• ????? ??? ???? ???? ?????? ????? ??? ??? ???
  ????? ???????????? ?? ??? ?????? .
• ????? ?????? ???????? .
• ??????? ?? ????????? ?????? ????? ????.
• ?? ??????? ?????? ?? ????? ????? ???????
  ???????????? ?? ????? ??????
• DNA Sequenceing .

8
??????? ????? ????? ??????? ????????

• ????? ??? ????? ?????? (DNA ) .
• ????? ????? ??????? ?? ???? ?? ??????? .
• ???? ???????? ????? ????? ?? ????? ???????
  ??????? ???????
• Human Genome Project
• ??????? ??????? ???? ???? ?? ???? ???? ?????? (
  ?????? ??????? ? ????? ???????? ? ????? ??????
  ...
• ????? ??????? ??????? Genotyping ??????? ?????? C
  B
• ????? ??????? ????????? ???????? .
• ????? ??????? ???????? HLA- tissuc typing ?? ????
  ????? ???????

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???? ??? ???????

• ???? ?????? ????? PCR ????? ????? ?????? ???????
  ????? ?????? DNA ????? ???????? ??????
• ???? ????? PCR ??? ????? ?????? ????? ?? ?????
  ?????? DNA? ??? ???????? ?? ????? ?? ????? ?????
  ???????? ?????? ??? ????? ????? ??? ???? ?????
  ??????? ????.
• ???? ?????? ????? PCR ????? ????? ?????? ???????
  ????? ?????? DNA ????? ???????? ??????.???? ???
  ??? ????????? ? ?? ?? ?? ???? ???? ????? ?????
  ??? ???

10
??????? ????? PCR

• 1- ???? ??????? ?? ???? ???? ????? ??????(DNA
  Sample ).

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??????? ????? PCR

• 2- ???????? (Primers)-
• ?????
• ????? (Forward).
• ???? ( Reverse).
• ??? ????? ?? ??????? ???????????? ?? ???? ????
  ????(20-25 bp) ???? ?????? ????? ?????? ??????
  ?? ????? ??????.

12
??????? ????? PCR

• 3-????? ??????? ( ( Hot Star Taq polymerase
  ??((Taq polymerase
• ?????? ?? ????? ??????? ???? Thermus aquaticus
  ???? ?????? ??????? ?? ???????? ????.
• ??????? ?????? ??????? ????????.
• ???? ?????? ?????? ?? 72º ?.

13
??????? ????? PCR

• 4- ??????? ???????????? ( Nitrogen Base dNTPs)-
• ????? Adenine
• ?????? Thymine
• ?????? Guanine
• ???????? Cytosine

dNTPs Deoxynuleoside triphosphates
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??????? ????? PCR

• 5- ????? ???? ( PCR Buffer10x).
• 6- ??? ?? ??????? ????? ????? ?????????? Mg2
  ???? ????? ???? ???? Cofactor ?????? ??????????
• 6- ??? ???? (DDW).

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??????? ????? PCR

• 7-???? ????? ??????? ????????(-(Thermocycler
• ???? ??? ?????? ????? ???? ??????? ???? ???? ?
  ???? ? ??????  ??? ???? ???? ??????? ??? ?? ?????
  ???????.

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????? ????? PCR???? ????? ?? ?????? ???????

• 1- ????? ??????? Denaturation ???????
• ??? ??? ???? ??????? ??? 94 ?? ???? ??? ?????
  ??????? ????? ?????? (DNA ) ????? .
• d.s. DNA ??? s.s.DNA
• 2- ????? ?????? ???????? Primers annealing
• ??? ??? ???? ??????? ??? ?? ??? 55-60 ?? ?????
  ???????? ????????? ????????? ?????? ???????
  ???????????? ?? ????? ?????? (DNA ) ????? .
• 3- ????? ???????? Extension
• ???? ???? ???? ??????? ??? 72 - 75 ? ????? ?????
  ??????? ????? ?? ???? ????? ?????? (DNA ) ??????
  . ?? ???? dNTPs
• ???? ??????? ?????? ???? ???? ????? ????? ????
  ????? ?????? (DNA ) ????? ?? ????? ? ?????? ????
  ???? ????? ?????? (DNA ) ??? ??? ??????? ???? ???
  .

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Figure 1 The different steps in PCR.
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Quantity of Reaction Master Mix
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Quantity of Reaction Master Mix

• ???? 23 ?????????? ?? ?????? ??????? Master Mix
  ??? ?? ????? ?? ?????? PCR ?? ???? 2 ??????????
  ?? ???? ????? (DNA) .
• ??? ???? ???????? ?? ???? ????? ??????? 3000 ????
  ?? ??????? ???? ????? ????? ???? ???? ???????
  ?????? ???? ????????.

20
???? ???????
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Figure3 Verification of the PCR product on gel
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????? ????? ??? PCR

• PCR?????? ?? ?? ???? Convential PCR ??? ?? ??
  ???? ??????? ???? ?? ??????? ???????.
• Real Time PCR ???? ????? ???? ??? ??? ??????
  ???? ?????? ?????? ???? ????? ?????? ????????
  ?????? ????? ??????? ???? ??????? ??? ?? ??????
  ???????? ???? ??? ????? ?????? (DNA ) ?????? ???
  ??? ???? ????? ?????????? ??? ???? ?????? ??? ???
  ???? ??? ???????? ????? ????? ???? ????? ???????
  ?? ?? ? ????? ????? ???? ?????? ??? ????? ???????
  ??????????????? .

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????? ??????? ????? ?????? ??? PCR

• ???? ????? ???? ????? ??? ????? ????? ?????? ??
  ????? ???? ??? .1 ??? ????????? Extraction
  sector.2 ??? ????? ???????Reagent preparation
  sector????? ??????? ?????? ????? (Pre PCR
  sector).3 ??? ??????? ????????? Amplification
  Detection sector????? ??? ????? ?????( Post- PCR
  Sector )

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A thermal cycler for PCR
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(No Transcript)
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(No Transcript)
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THANKSforLISTINING