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Pcr
•



.
               •
      (DNA )

    (DNA )
DNA
Kary Mullis
           DNA
PCR
                        •
in vitro         DNA
       Thermal Cycler

                        •

                        •

                        •
Pcr
Pcr
•


                  •
                  •


 .

                  •

                  •
DNA Sequenceing   •
(DNA )        •
                 .                   •
                                     •
Human Genome Project
                                     •

      C&B              Genotyping    •
                                     •
                HLA- tissuc typing   •
PCR         •
     DNA
        PCR     •
          DNA


    PCR         •
     DNA
:
PCR
DNA             - •
            Sample
PCR


      - Primers       -   •
                          •
           Forward        •
           Reverse        •
                          •
           20-25 bp
PCR
( Hot Star Taq polymerase            -   •
                    :(Taq polymerase
 Thermus aquaticus                       •


                                         •

                     º                   •
PCR
 - Nitrogen Base dNTPs                -   •
                      Adenine             
                     Thymine              
                     Guanine              
                     Cytosine             




dNTPs :Deoxynuleoside triphosphates
PCR
       PCR Buffer10x        -   •
Mg+2                        -   •
                 Cofactor




                 DDW        -   •
PCR
-:(Thermocycler   -   •
PCR

                                    Denaturation              -
     .   (DNA )
                                             s.s.DNA   d.s. DNA

                        :Primers annealing                    -
                              -
                          .     (DNA )

                                        :Extension            -
(DNA )                              -
                                             dNTPs        .

              (DNA )
                               (DNA )
Figure 1: The different steps in PCR.
Quantity of Reaction Master Mix
Quantity of Reaction Master Mix
Master Mix                         
               PCR
                      DNA

                                   
Pcr
Figure3 : Verification of the PCR
         product on gel
PCR
 Convential PCR               PCR


                  : Real Time PCR

                          (DNA )

                      .
PCR
                                           •

              Extraction sector      .1
Reagent preparation sector           .2
        (Pre – PCR sector)
Amplification + Detection             .3
                                  sector
           ( Post- PCR Sector )
A thermal cycler for PCR
Pcr
Pcr
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    for
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Pcr

  • 2. • . • (DNA ) (DNA )
  • 4. PCR • in vitro DNA Thermal Cycler • • •
  • 7. • • . • • DNA Sequenceing •
  • 8. (DNA ) • . • • Human Genome Project • C&B Genotyping • • HLA- tissuc typing •
  • 9. PCR • DNA PCR • DNA PCR • DNA :
  • 10. PCR DNA - • Sample
  • 11. PCR - Primers - • • Forward • Reverse • • 20-25 bp
  • 12. PCR ( Hot Star Taq polymerase - • :(Taq polymerase Thermus aquaticus • • º •
  • 13. PCR - Nitrogen Base dNTPs - • Adenine  Thymine  Guanine  Cytosine  dNTPs :Deoxynuleoside triphosphates
  • 14. PCR PCR Buffer10x - • Mg+2 - • Cofactor DDW - •
  • 16. PCR Denaturation - . (DNA ) s.s.DNA d.s. DNA :Primers annealing - - . (DNA ) :Extension - (DNA ) - dNTPs . (DNA ) (DNA )
  • 17. Figure 1: The different steps in PCR.
  • 18. Quantity of Reaction Master Mix
  • 19. Quantity of Reaction Master Mix Master Mix  PCR DNA 
  • 21. Figure3 : Verification of the PCR product on gel
  • 22. PCR Convential PCR PCR : Real Time PCR (DNA ) .
  • 23. PCR • Extraction sector .1 Reagent preparation sector .2 (Pre – PCR sector) Amplification + Detection .3 sector ( Post- PCR Sector )
  • 24. A thermal cycler for PCR
  • 27. THANKS for LISTINING

Notas del editor

  1. البادئات Primers وهي عبارة عن نيوكلوتيدات قليلة( Oligonucleotides (18 – 20 أساس آزوتي قادرة على الارتباط مع الأسس الآزوتية للحمض النووي المراد تضخيمه، وذلك في منطقة ذات ترتيب مميز ونوعي غير متبدل للأسس الازوتية في الحمض النووي أو ما يعرف بمنطقة عالية الحفظ Highly Conserved Region .كميات وافرة من النيوكليوزيدات ثلاثية الفوسفات منقوصة الأوكسجين(dATP,dCTP,dGTP,dTTP)Deoxynuleoside triphosphates(dNTPs)أنظيم البوليميراز Polymerase مقاوم للحرارة المرتفعة، وأهمهاTaq Polymerase المستخلص من بكتيريا تعيش في الينابيع الحارةThermusaquaticus.محاليل واقيه Buffersشوا رد مناسبة، أهمها شاردة المغنيزيوم Mg+2 التي تعتبر عامل متمم Cofactor لأنظيم البوليمراز.
  2. 1. جهاز للتحكم بدرجات حرارة التفاعل بشمل دقيق و متتالي ( الدورة الحراريةThermocycle ) : ويقوم هذا الجهاز بتغير درجة الحرارة بشكل سريع ، لآن تغير درجة الحرارة هو الأساس الذي تقوم عليه فكرة هذه التقنية .2. البليمريز : وهو الإنزيم الذي يقوم ببناء وترتيب القواعد النيتروجينية ( حدات الحمض النووي (DNA ) ) ، ويجب أن يكون هذا الإنزيم مقاوم للحرارة العالية ليتمكن من العمل . و قد اكتشف انزيم مقاوم للحرارة و اسم تاج Tag3. مجموعة متفرقة من القواعد النيتروجينية: ( A T C G ) ليتمكن الإنزيم من ترتيبها في مواقعها أثناء عملية نسخ الحمض النووي (DNA ) .4. بريمر Primer : وهو قطعة صغيرة من الحمض النووي (DNA ) ليتمكن الإنزيم من بداء البناء و النسخ عليها .5. والشيء الأهم هو وجود نسخة من الحمض النووي (DNA ) المراد نسخه .6. بالإضافة إلى محلول أو وسط ليتم به التفاعل : وهذا المحلول يختلف بين تفاعل و أخر .