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Conventional , qPCR & RTPCR

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Mohammed Bahgat Mohammed Sofyan
Mohammed Bahgat Mohammed Sofyan

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Education
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PCR, qPCR & RTPCR BY dr: Mohammed Bahgat Mohammed Sofyan Assistant lecturer of medical biochemistry, Faculty of medicine, Al-Azhar university (Assiut branch) 1
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‫صورة‬ ‫كل‬ ‫على‬ ‫اضغط‬ ‫الشاش‬ ‫عرض‬ ‫وضع‬ ‫في‬ ‫ة‬ ‫قنواتنا‬ ‫على‬ ‫وادعمنا‬ ‫اإلنترنت‬ ‫على‬
‫على‬ ‫ملفي‬ ‫في‬ ‫هنا‬ ‫أرفعها‬ ‫التي‬ ‫شرائحي‬ ‫ومذاكرة‬ ‫بالتعلم‬ ‫وأسعد‬ ‫وأرحب‬ ‫أسمح‬ Slide share website ‫وال‬ ‫مانع‬ ‫أيضا‬ ‫من‬ ‫نسخ‬ ‫شريحة‬ ‫او‬ ‫اثنين‬ ‫عند‬ ‫الضرورة‬ ، ‫وال‬ ‫مانع‬ ‫من‬ ‫شرح‬ ‫البورب‬ ‫وينت‬ ‫الخاصة‬ ‫بي‬ ‫للغير‬ ‫بشرط‬ ‫عدم‬ ‫إزالة‬ ‫اسمي‬ ‫من‬ ‫البوربوينت‬ ، ‫فال‬ ‫أسمح‬ ‫أبدا‬ ‫بإزال‬ ‫ة‬ ‫اسمي‬ ‫من‬ ‫على‬ ‫الباوربوينت‬ ‫ووضع‬ ‫اسمك‬ ‫بدال‬ ‫منه‬ ‫لتصبح‬ ‫وكأنك‬ ‫من‬ ‫صممتها‬ ‫فهذه‬ ‫سرقة‬ ‫ال‬ ‫أسمح‬ ‫بها‬ ‫وتضييع‬ ‫لحق‬ ‫من‬ ‫تعب‬ ‫في‬ ‫عملها‬ . ‫وفقكم‬ ‫هللا‬ ‫وإياي‬ ‫للتعلم‬ ‫ونفع‬ ‫اآلخرين‬ I allow, welcome, and be happy to learn and study my slides that I upload here in my profile on Slide share website There is also no objection to copying one or two slides when necessary, and there is no objection to explaining my PowerPoint to others on the condition that my name is not removed from the PowerPoint. I never allow my name to be removed from PowerPoint and to replace it with yours, Make it look like you designed it. This is theft that I do not allow and a waste of the right of those who are tired in this work. May God bless you and me for learning and benefiting others 5
Conventional PCR Conventional PCR : ‫ال‬ ‫كل‬ ‫علي‬ ‫حصلت‬ ‫ما‬ ‫بعد‬ DNA ‫ال‬ ‫من‬ DNA extraction ‫أعمل‬ ‫الزم‬ ‫يبقي‬ ‫منه‬ ‫جزء‬ ‫هستخدم‬ ‫كله‬ ‫هستخدمه‬ ‫مش‬ ‫طبعا‬ PCR ‫ال‬ ‫ندخل‬ ‫نقوم‬ DNA ‫ال‬ ‫علي‬ PCR PCR is in-vitro amplification of specific DNA sequences ( amplicon ). 6
‫ال‬ PCR ‫من‬ ‫خليط‬ ‫عن‬ ‫عبارة‬ ‫ده‬ 1- Master mix ➡ thermostable Tag polymerase, deoxynucleotides, Mg, buffer 2- two oligonucleotide primer 3- DNA sample 4- DNAs RNAs free water ‫الخطوات‬ ‫يحدث‬ ‫معينة‬ ‫بنسب‬ ‫بعضه‬ ‫علي‬ ‫كله‬ ‫نضع‬ ‫التالية‬ 7
8
Polymerase Chain Reaction 9
Denaturation, Anneling and Extention 10
A. Double strand DNA B. Denature 96º 50º C. Anneal primers 50º D. Polymerase binds 72º Taq Taq 11
72º Taq Taq E. Copy strands 1 2 3 4 F. Denature 96º First round of cDNA synthesis (4 strands) Taq Taq 12
1 2 3 4 50º G. Anneal primers 13
1 2 3 4 Taq Taq Taq Taq 72º H. Polymerase binds 14
1 2 3 4 Taq Taq Taq Taq I. Copy strands 72º Second round of cDNA synthesis (8 strands) 15
1 2 3 4 J. Denature at 96º Anneal primers at 50º 16
1 2 3 4 72º K. Bind polymerase (not shown) and copy strands Third round of cDNA synthesis (16 strands) 17
1 2 3 4 L. Denature at 96º Anneal primers at 50º 18
1 2 3 4 M. Copy strands at 72º Fourth round of cDNA synthesis (32 strands) 72º 19
1 2 3 4 cDNA strands (32) are now shown as lines 20
1 2 3 4 After 5 rounds there are 32 double strands of which 24 (75%) are are same size 21
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STEPS OF PCR 0_ hot start PCR ➡: This step is only required for DNA polymerases to be activated and heating the reaction chamber to a temperature of 94–96 °C . It takes 3_9 minutes. 1_ denaturation ➡ the mixture is heated to 95 *C for 30 seconds to separate 2 strands of DNA. 2_ annealing ➡ the mixture is cooled to 50*C to allow the 2 primers to bind to the 2 strands if the target DNA for 20–40 seconds, A typical annealing temperature is about 3–5 °C below the Tm of the primers used. 3_ elongation ➡the mixture is heated to 72* C for 1_2 minutes to allow polymerase to elongate each primer by copying the single strand template. The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify. 4_ cycles are repeated 30_35 cycles from step 1_3 5_ Final elongation: This single step is optional, but is performed at a temperature of 70– 74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated. 6_ Final hold: The final step cols the reaction chamber to 4–15 °C for an indefinite time, and maybe employed for short-term storage of the PCR products. 23
After Conventional PCR ‫فايدة‬ ‫ال‬ Conventional PCR ‫انه‬ ‫بيكبرلي‬ ‫الجزء‬ ‫اللي‬ ‫انا‬ ‫عاوز‬ ‫أشتغل‬ ‫عليه‬ ‫ممكن‬ ‫لو‬ ‫جريته‬ ‫علي‬ gel electrophoresis ‫اعرف‬ ‫هل‬ ‫حصل‬ ‫تكبير‬ ‫وال‬ ‫ايه؟‬ Amplified products can be detected only at the end of the PCR reaction by doing agarose gel electroAphoresis. ‫بعد‬ ‫كده‬ ‫ممكن‬ ‫اشتغل‬ ‫بيه‬ RFLP to detect any mutations. ‫ممكن‬ ‫اعمل‬ ‫بيه‬ DNA sequencing and Fragment analysis. ‫ممكن‬ ‫يأخذ‬ ‫ونفعل‬ ‫به‬ ‫ما‬ ‫نريد‬ . 24
Revers transcriptase ‫واخد‬ ‫أنا‬ ‫لو‬ RNA ‫ال‬ ‫جهاز‬ ‫في‬ ‫أكبره‬ ‫وعاوز‬ Conventional PCR ‫ال‬ ‫هدخل‬ ‫بس‬ ،‫الماضي‬ ‫زي‬ ‫هيبقي‬ RNA ‫له‬ ‫وأضيف‬ ‫الجهاز‬ ‫علي‬ Revers transcriptase and RNAs inhibitors ‫حوالي‬ ‫وأتركهم‬ About 1 hours in 48 *C ‫ال‬ ‫هيتحول‬ RNA ‫إلي‬ cDNA ‫الماضية‬ ‫الخطوات‬ ‫نفس‬ ‫بقه‬ ‫أعمل‬ 25
Agarose gel electrophoresis ‫علي‬ ‫أحصل‬ ‫ما‬ ‫بعد‬ PCR product ‫علي‬ ‫أجريه‬ ‫إيه؟‬ ‫وال‬ ‫للجين‬ ‫تكبير‬ ‫حصل‬ ‫أشوف‬ ‫عاوز‬ Agarose gel electrophoresis ‫من‬ ‫معينة‬ ‫بتركيزات‬ ‫بيتعمل‬ ‫ده‬ Agrose and EDTA buffer ‫عليها‬ ‫ونضع‬ ،‫اآلن‬ ‫لذكرها‬ ‫داعي‬ ‫ال‬ ‫وخالفه‬ ethidium bromide ‫ال‬ ‫يظهر‬ ‫لكي‬ DNA ‫ال‬ ‫لمبة‬ ‫تحت‬ UV ‫في‬ ‫أحطه‬ ،‫الجيل‬ ‫يتعمل‬ ‫لما‬ track ‫وأعمل‬ load ‫ال‬ ‫للعينات‬ DNA ‫ال‬ ‫وضع‬ ‫وعند‬ ‫سالبة‬ ‫هيا‬ ‫اللي‬ buffer ‫ال‬ ‫يتحرك‬ ‫الكهربائي‬ ‫التيار‬ ‫وتوصيل‬ DNA ‫حسب‬ ‫علي‬ ‫للموجب‬ ‫السالب‬ ‫القطب‬ ‫من‬ Molecular weight and charge ‫فيه‬ ‫لو‬ ‫طبعا‬ DNA ‫خالل‬ ‫ومن‬ ‫فين‬ ‫هيتحرك‬ ‫هشوف‬ ‫معين‬ ‫جين‬ ‫فيه‬ ‫ولو‬ ،‫لألمام‬ ‫هيتحرك‬ ladder ‫كم‬ ‫علي‬ ‫يحتوي‬ ‫الجين‬ ‫هذا‬ ‫حساب‬ ‫يتم‬ ‫معين‬ base pair. Amplified products can be detected only at the end of the PCR reaction by doing agarose gel electrophoresis. To approximate the size of the PCR product by using a ladder. 3_ to calculate Tm is about 4 (G _ T) + 2 (A _ T) ‫أخري‬ ‫حساب‬ ‫طرق‬ ‫ولها‬ 4- Ramp time : It's the time that the thermal cycler needed to change from one temperature to the next. 26
DNA analysis by agarose gel electrophoresis. 27
28
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30 Principle of PCR (video)
Principle of qPCR 31
Defintion of qPCR Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses. 32
Real time PCR ( qPCR ) ‫ال‬ PCR ‫من‬ ‫خليط‬ ‫عن‬ ‫عبارة‬ ‫ده‬ : 1-Master mix ➡ thermostable Tag polymerase, deoxynucleotides, Mg, buffer 2- two oligonucleotide primer 3- DNA sample 4- DNAs RNAs free water 5- taqman probe or SYBER green 6- internal positive control and it's primer. ‫معينة‬ ‫بنسب‬ ‫بعضه‬ ‫علي‬ ‫كله‬ ‫نضع‬ , ‫ال‬ ‫مثل‬ ‫خطوات‬ ‫يحدث‬ ‫ذلك‬ ‫بعد‬ Conventional PCR ‫هللا‬ ‫شاء‬ ‫ان‬ ‫الدرس‬ ‫هذا‬ ‫في‬ ‫عنها‬ ‫سنتحدث‬ ‫قليلة‬ ‫زيادات‬ ‫مع‬ 33
Principle Of real time PCR 1- as conventional PCR in addition to ‫مثل‬ ‫ال‬ PCR ‫العادي‬ ‫بالضبط‬ ‫بس‬ ‫فيه‬ ‫زيادة‬ ‫الزيادة‬ ‫إن‬ ‫فيه‬ ‫شيئ‬ ‫بيحسب‬ ‫ويعد‬ ‫هذه‬ ‫الدورات‬ ‫أثناء‬ ‫التضاعف‬ ‫وليس‬ ‫ف‬ ‫ي‬ ‫نهايته‬ It monitors the amplification of targeted DNA molecule during the PCR and not at its end ‫هذه‬ ‫الزيادة‬ ‫هي‬ 34
2_ Optical module ( to detect the fluorescence in the tubes during the run) As : A_ taqman probe ‫ده‬ ‫بيبقي‬ ‫عليه‬ flourophre as VIC and FAM ‫ودول‬ ‫بيشعوا‬ ‫ضوء‬ fluorescence ، ‫معاهم‬ ‫ال‬ Quencher ‫بيمنع‬ ‫أو‬ ‫يمتص‬ ‫هذا‬ ،‫اإلشعاع‬ ‫لو‬ ‫حصل‬ ‫ووجد‬ ‫ال‬ DNA ‫المراد‬ ‫قياسه‬ ‫هيحصل‬ elongation by taq polymerase ‫يقوم‬ ‫يكسر‬ ‫هذا‬ ‫ال‬ taqman probe ‫يقوم‬ ‫ال‬ Quencher ‫يبعد‬ ‫عن‬ ‫ال‬ flourophre ‫يقوم‬ ‫يحصل‬ ‫اشعاع‬ ‫ويتم‬ ‫التقاطه‬ ‫ب‬ detector 35 Principle Of real time PCR
‫اذا‬ ‫لو‬ ‫فيه‬ cycle ،‫اتعملت‬ ‫يطلع‬ ‫اشعاع‬ ‫ولو‬ ‫مفيش‬ ‫ال‬ ‫يوجد‬ ،‫اشعاع‬ ‫وبكده‬ ‫يعرف‬ ‫عدد‬ ‫ال‬ cycles ‫عن‬ ‫طريق‬ ‫فالتر‬ ‫بتعمل‬ Detection to this Flourescence light ‫ويرسم‬ ،‫منحني‬ ‫وبكده‬ ‫يقدر‬ ‫يعمل‬ Quantitative assay to any DNA or RNA ‫بعدد‬ ‫الضوء‬ ‫الخارج‬ Amount of Flourescence detected by optical filters. ‫يعني‬ ‫كل‬ ‫ما‬ ‫يحصل‬ ‫دورة‬ ،‫وتضاعف‬ ‫يزداد‬ ‫عدد‬ ‫الضوء‬ ‫الخارج‬ . ‫هيحصل‬ ‫عندي‬ ‫اآلن‬ ‫ثالث‬ ‫احتماالت‬ : 36 Principle Of real time PCR
‫اإلحتمال‬ ‫األول‬ ⬅ ‫لو‬ ‫مفيش‬ ‫أصال‬ DNA ‫مش‬ ‫هيركب‬ ‫ال‬ taqman ‫علي‬ ‫ال‬ DNA ‫مطلقا‬ ‫وبالتالي‬ ‫مش‬ ‫هيطلع‬ ‫ضوء‬ ‫مطلقا‬ ‫وتكون‬ ‫النتيجة‬ ‫سلبية‬ . ‫اإلحتمال‬ ‫الثاني‬ : ‫أن‬ ‫يكون‬ ‫عدد‬ ‫الفيروسات‬ ‫قليل‬ ‫مثال‬ 5 ( ‫مثال‬ ‫افتراضي‬ ‫غير‬ ‫صحيح‬ ) ‫بعد‬ ‫الدورة‬ ‫األولي‬ ‫سيصب‬ ‫ح‬ 10 ‫ينتج‬ ‫عنهم‬ ‫مثال‬ 10 Flourescence ، ‫بعد‬ ‫الثانية‬ 20 ، ‫بعد‬ ‫الثالثة‬ 40 ، ‫بعد‬ ‫الرابعة‬ 80 ‫بعد‬، ‫الخامسة‬ 160 ، ‫بعد‬ ‫السادسة‬ 320 ‫بعد‬، ‫السابعة‬ 640 ‫بعد‬، ‫الثامنة‬ 1280 ، ....... ‫وهكذا‬ ‫يبقي‬ ‫طلع‬ 1280 ‫ضوء‬ ‫بعد‬ 8 ‫دورات‬ ‫اإلحتمال‬ ‫الثالث‬ : ‫أن‬ ‫يكون‬ ‫عدد‬ ‫الفيروسات‬ ‫كبير‬ ‫مثال‬ 100 ( ‫مثال‬ ‫افتراضي‬ ‫غير‬ ‫صحيح‬ ) ‫بعد‬ ‫الدورة‬ ‫األولي‬ ‫هيكون‬ 200 ‫ينتج‬ ‫عنهم‬ ‫مثال‬ 200 Flourescence ، ‫بعد‬ ‫الثانية‬ 400 ، ‫بعد‬ ‫الثالثة‬ 800 ‫بعد‬، ‫الرابعة‬ 1600 ، ....... ‫وهكذا‬ ‫يبقي‬ ‫طلع‬ 1600 ‫ضوء‬ ‫بعد‬ 4 ‫دورات‬ ‫فقط‬ ‫طبعا‬ ‫للحصول‬ ‫علي‬ ‫مليار‬ ‫نسخة‬ ‫هنحصل‬ ‫عليهم‬ ‫في‬ ،‫الحالتين‬ ‫ولكن‬ ‫األكثر‬ ‫في‬ ‫عدد‬ ‫الفيروسات‬ ‫من‬ ‫البداية‬ ‫هو‬، ‫اللي‬ ‫هيوصلهم‬ ‫في‬ ‫عدد‬ ‫دورات‬ ‫أقل‬ 37 Principle Of real time PCR
‫العلماء‬ ‫قالوا‬ ،‫خالص‬ ‫ممكن‬ ‫مثال‬ ‫نحط‬ threshold ‫وليكن‬ ‫مثال‬ 1200 Flourescence ‫ونشوف‬ ‫مين‬ ‫اللي‬ ‫هيجيبهم‬ ‫في‬ ‫عدد‬ ‫دورات‬ ،‫أقل‬ ‫يعني‬ ‫مين‬ ‫اللي‬ ‫هيطلع‬ ‫ضوء‬ ‫معين‬ ( threshold) ‫في‬ ‫عدد‬ ‫دورات‬ ،‫أقل‬ ‫وسموا‬ ‫هذا‬ ‫ال‬ Ct ➡ cycle threshold Ct: cycle numbers at which the fluorescence signals is detected by instrument significantly (i.e. passed fixed threshold) ‫طبعا‬ ‫كل‬ ‫ما‬ ‫حصلنا‬ ‫علي‬ ‫هذا‬ ‫الضوء‬ threshold ( ex ➡ 1200 fluorescence) ‫في‬ ‫عدد‬ ‫دورات‬ ‫أقل‬ ‫يبقي‬ ‫كده‬ ‫بادئين‬ ‫بنسخ‬ ‫كبيرة‬ ‫من‬ ‫الفيروس‬ So real time PCR can do accurate calculation of initial copy number of templete. ‫يعني‬ ‫في‬ ‫المثال‬ ‫اللي‬ ‫معانا‬ ‫في‬ ‫اإلحتمال‬ ‫الثاني‬ ‫وصل‬ ‫لل‬ 1200 ‫ضوء‬ ‫بعد‬ 8 ،‫دورات‬ ‫في‬ ‫حين‬ ‫اإلحتمال‬ ‫الثالث‬ ‫وصل‬ ‫لل‬ 1200 ‫بعد‬ 4 ‫دورات‬ ‫فقط‬ ‫يبقي‬ ‫في‬ ‫اإلحتمال‬ ‫األول‬ CT ➡ 8 ‫وفي‬ ‫اإلحتمال‬ ‫الثاني‬ CT ➡ 4 ‫وبالتالي‬ ‫اللي‬ ‫وصل‬ ‫لل‬ threshold ‫في‬ ‫عدد‬ ‫دورات‬ ‫أقل‬ ‫يبقي‬ ‫هو‬ ‫اللي‬ ‫كان‬ ‫في‬ ‫األول‬ ‫أكثر‬ .... ‫وهكذا‬ . ‫طبعا‬ ‫السلبي‬ ‫لن‬ ‫يخرج‬ ‫ضوءا‬ ‫مطلقا‬ . ‫وعن‬ ‫طريق‬ ‫عديد‬ ‫من‬ ‫ال‬ standers ‫معلومي‬ ‫التركيز‬ ‫أحسب‬ ‫لهم‬ Ct ‫ومن‬ ‫خالل‬ ‫ذلك‬ ‫أعمل‬ Stander curve ‫وأحسب‬ ‫ال‬ unknown. 38 Principle Of real time PCR
Principle Of real time PCR Quantitative PCR relies on the principal that the quantity of target at the start of the reaction is proportional to amount of product produced during the exponential phase. 39
40
Detection of qPCR • Ct values are directly related to the starting quantity of DNA 41
Standard curve. 42
‫فيه‬ ‫مادة‬ ‫مشعة‬ ‫أخري‬ ‫تستعمل‬ ‫في‬ qPCR ‫وهي‬ B_ SYBR Green It's non-specific, It binds to double-stranded DNA formed during the PCR. It fluoresces when bound to double-stranded DNA and detected by a detector. ‫مادة‬ ‫بتمسك‬ ‫في‬ ‫ال‬ double-stranded DNA ‫كل‬ ‫ما‬ ‫زاد‬ ‫ال‬ double-stranded DNA ‫زاد‬ ‫إمساك‬ ‫ال‬ SYBR Green with double-stranded DNA ‫كل‬ ‫ما‬ ‫زاد‬ ‫اإلشعاع‬ . ‫طبعا‬ ‫كل‬ ‫ما‬ ‫زاد‬ ‫عدد‬ ‫ال‬ DNA ‫من‬ ‫البداية‬ ‫يزداد‬ ‫الضوء‬ ‫الناتج‬ ‫ال‬ FlourescenceFluorescence ‫مثل‬ ‫الماضي‬ ‫تماما‬ ‫فيه‬ ‫طرق‬ ‫أخري‬ ‫غير‬ ‫ال‬ Taqman and SYBR Green ‫لكن‬ ‫غير‬ ‫مشهورة‬ 43 Principle Of real time PCR
Revers transcriptase ‫واخد‬ ‫أنا‬ ‫لو‬ RNA ‫ال‬ ‫جهاز‬ ‫في‬ ‫أكبره‬ ‫وعاوز‬ Conventional PCR ‫ال‬ ‫هدخل‬ ‫بس‬ ،‫الماضي‬ ‫زي‬ ‫هيبقي‬ RNA ‫له‬ ‫وأضيف‬ ‫الجهاز‬ ‫علي‬ Revers transcriptase and RNAs inhibitors ‫حوالي‬ ‫وأتركهم‬ About 1 hours in 48 *C ‫ال‬ ‫هيتحول‬ RNA ‫إلي‬ cDNA ‫الماضية‬ ‫الخطوات‬ ‫نفس‬ ‫بقه‬ ‫أعمل‬ 44
Revers transcriptase 2 _ ‫لو‬ ‫أنا‬ ‫بعمل‬ quantification ‫ل‬ RNA ‫ممكن‬ ‫يكسر‬ ‫مني‬ ‫أثناء‬ ،‫استخالصه‬ ‫خاصة‬ ‫إنه‬ ‫سهل‬ ،‫التكسير‬ ‫فتطلع‬ ‫النت‬ ‫يجة‬ ‫سلبية‬ ‫نتيجة‬ ‫إن‬ ‫شغلي‬ ‫خطأ‬ ‫والصواب‬ ‫إن‬ ‫النتيجة‬ ‫ليست‬ ،‫كذلك‬ ‫قالك‬ ‫خالص‬ ‫نعمل‬ Internal positive control ‫داخل‬ ‫كل‬ ‫عينة‬ ‫يتم‬ ‫وضع‬ ‫عينة‬ ‫إيجابية‬ ‫ولها‬ Specific primer, specific probe ‫ونقيسها‬ ‫الزم‬ ‫تعطي‬ ،‫ايجابية‬ ‫وتكون‬ ‫في‬ ‫جميع‬ ‫العينات‬ ‫نفس‬ ،‫القيمة‬ ‫وهنا‬ ‫نحكم‬ ‫علي‬ ‫العينة‬ ‫إنها‬ ‫سلبية‬ ‫في‬ ‫حالة‬ ‫واحدة‬ ‫إذا‬ ‫طلعت‬ ‫الحالة‬ ‫سلبية‬ ‫وال‬ Internal positive control is positive. 3 - ‫في‬ ‫حالة‬ ‫ال‬ Gene expression analysis : Different samples maybe have different numbers of cells from the start, So PCR solution reaction will be done for the target gene and reference gene, Ct value of the target gene will be normalized with the Ct value of the reference gene. 2*- 🔼 🔼 Ct method will be utilized to find the level of gene expression levels. 45 Revers transcriptase
46
Application of real type PCR ( qPCR ) 1_ quantitative assay of bacteria, viruses, and parasites 2_ quantitative detection of genes, RNA, DNA, and plasmid 3_ quantitative assay of gene expression 4_ quantitative detection of SNP # ‫دمحمد‬ _ ‫بهجت‬ _ ‫سفيان‬ _ medical_biochimestry 47
For Allele 1 - only VIC™ dye signal is generated SNP scoring assay UsingMGB probes G C A C NFQ FAM VIC G T A T FAM VIC For Allele 2 - only FAM™ dye signal is generated NFQ NFQ NFQ 48
For Allele 1 - only VIC™ dye signal is generated For Allele 2 - only FAM™ dye signal is generated SNP scoring assay UsingMGB probes G C A C NFQ FAM VIC G T A T FAM VIC NFQ NFQ NFQ 49
Principle of qPCR 50
51
REFERENCES : • _ https://labtestsonline.org • _ https://www.medscape.com • _ https://www.wikipedia.org • _ https ps://www.labcorp.com • _ https://www.uptodate.com • _ https://www.ncbi.nlm.nih.gov Home - PubMed – NCBI • _TIETZ TEXTBOOK OF CLINICAL CHEMISTRY AND MOLECULAR DIAGNOSTICS, SIXTH EDITION 2018. • _Essential of clinical pathology book; 1st edition; Shirish M Kawthalkar; 2010. • _Essential of biochemistry book ;1st edition; 2012. • _ ABC of clinical hematology ; 4th edition ; Drew Provan; 2018. • _ Harper's illustrated biochemistry 30th edition 2015. • _ Lippincott's illustrated review of biochemistry sixth edition 2014. • _ Lecturea Notes Clinical Biochemistry, 9th Edition Walker, Simon, 2103. • _Some audios and videos from Well-known, trusted professors who study from accredited books. 52
‫اليوتيوب‬ ‫علي‬ ‫البحث‬ ‫أو‬ ،‫اليوتيوب‬ ‫علي‬ ‫قناتي‬ ‫دي‬ ‫د‬ ‫باسم‬ . ‫سفيان‬ ‫بهجت‬ ‫محمد‬ ، ‫وتشتركو‬ ‫تتابعوني‬ ‫ياريت‬ ‫حتى‬ ‫بالقناة‬ ‫ا‬ ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ https://www.youtube.com/channel/UCaYs1d8s0ntZvteHS3mMmGA ‫ب‬ ‫الفيس‬ ‫في‬ ‫البحث‬ ‫أو‬ ،‫انضمامكم‬ ‫يشرفنا‬ ‫الفيسبوك‬ ‫علي‬ ‫بتاعتي‬ ‫الصفحة‬ ‫دي‬ DMBMS2018 ، ‫حتى‬ ‫تتابعوني‬ ‫ياريت‬ ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ https://www.facebook.com/DMBMS2018/ ‫باسم‬ ‫التلجرام‬ ‫علي‬ ‫قناتي‬ ‫ودي‬ ِ ‫للناس‬ ‫أنفعهم‬ ‫الناس‬ ‫خير‬ 📚 ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ِريت‬‫ا‬‫ي‬ https://t.me/DMBMS2020 ‫على‬ ‫بروفايلي‬ ‫لينكيدإن‬ ‫وتعملولي‬ ‫تتابعوني‬ ‫ياريت‬ endorsement ‫مهاراتي‬ ‫على‬ https://www.linkedin.com/in/mohammed-bahgat-sofyan-8ba012142/ ‫موقع‬ ‫على‬ ‫بروفايلي‬ ‫وده‬ SlideShare ‫تتابعونا‬ ‫ياريت‬ https://www.slideshare.net/MohammedBahgatMohamm1 ‫وده‬ ‫بروفايلي‬ ‫الشخصي‬ ‫الفيسبوك‬ ‫علي‬ https://www.facebook.com/mohammed.bahgat.165 ‫الفيسبوك‬ ‫على‬ ‫بنا‬ ‫الخاص‬ ‫اللوجوا‬ ‫وده‬ # ‫دمحمد‬ _ ‫بهجت‬ _ ‫سفيان‬ _ medical_biochimestry 53
T H A N K Y O U M E E T O N T H E B E S T L A T E R , G O D W I L L I N G 54

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