4- pcr types.pptxMolecular geatics PCR Types Basic scince

Molecular geatics PCR Types Basic scince

1. Molecular Genetics Lecture 4 PCR types

2. Colony PCR Nested PCR Multiplex PCR Long PCR Gradient PCR Hot Start PCR Inverse PCR Overlapping PCR DNA fingerprinting RAPD AFLP RFLP etc.. Reverse Transcriptase PCR (RT-PCR) Real Time PCR ( qRT -PCR) بعض الانواع الخاصة لتفاعل البلمرة المتسلسل PCR types

3. salts (ions) dNTPs Primers DNA Polymerase Template DNA Conventional PCR

4. Colony PCR

5. Nested PCR

6. Multiplex PCR

7. Gradient PCR

8. Hot Start PCR Used to optimize the yield of the desired amplified product in PCR and simultaneously to suppress nonspecific amplification. hotstart normal

9. Long PCR Standerd PCR Long PCR 200 bp – 2K bp 5K -20K Alvin Submersible for Exploration of Deep Sea Vents 95ºC 30 S 30-60 ºC 30 S 72 ºC 7min

10. Inverse PCR عزل الـ DNA الكلي من الخلية تقطيع الـ DNA بواسطه احد انزيمات القطع الذي يؤدي الي تكوين مقاطع من الـ DNA لها نفس النهايات الزجة ( Stecky Ends ). اضافة انزيم الـ ligase الذي يربط هذه النهايات ببعضها مكونة مقاطع دائرية من الـ DNA يتم استخدام انزيم قطع جديد بحيث ان يقوم بالقطع في المنطقة معروفة التتابع مما يؤدي الي انتقال هذه المنطقة المعروقه الي اطراف المقطع تستخدم هذه القطع كقالب اثناء تفاعل الـ PCR بحيث ترتبط البرايمرز في المنطقه معروفه التتابع التي توجد علي الاطراف يتم تحديد التتابع الخاص بهذا المقطع بواسطة الـ DNA sequencer

11. when is a “primer” a primer? 5’ 3’ 5’ 5’ 5’ 3’ 3’ 3’

12. Overlapping PCR PCR 1 PCR 2 يتم خلط نواتج كل من الـ PCR1 و الـ PCR2 في انبوبه جديدة و يتم استخدامهما كقالب للـ DNA في الـ PCR3 PCR 3

13. AFLP RAPD SSR ISSR RFLP MASAP VNTR CAPS SRAP SSCP SNP COS DArT SCAR RDA SAMPL Molecular Markers (DNA fingerprinting)

14. Restriction Fragment Length Polymorphism (RFLP)

15. Restriction Fragment Length Polymorphism (RFLP) Restriction site? Restriction Enzymes: E.g., EcoR1

16. Randomly amplified polymorphic DNA RAPD

17. Amplified Fragment Length Polymorphism AFLP-PCR

18. Reverse transcription

19. qRT-PCR is used to qualitatively detect gene expression Why RT-PCR? Gene expression analysis Cancer research Drug research Disease diagnosis and management Viral quantification Food testing Percent GMO food Animal and plant breeding Gene copy number

20. RNA AAAAAAAAAAAA TTTTTT oligo dT primer All messenger RNAs (mRNA) have a poly A tail The RT reaction can be primed by a: target specific primer (i.e. primer targeting VHSV nucleocapsid (N) gene) oligo dT primer (a primer consisting of a run of T’s that targets the mRNA poly A tail) random primers (a mix of 6 base primers consisting of random nucleotides)

21. RT-PCR mRNA oligo dT RT: Reverse transcriptase cDNA mRNA 5´ 3´ 5´ 3´ Templat for RT- PCR

22. Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses. 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 ∆ Fluorescence C T C T Greater starting target Less starting target < Real-Time PCR

23. How does PCR work? Log Target DNA Cycle Number Theoretical

24. Conventional PCR problem Log Target Agarose gel electrophoresis following PCR Cycle Number

25. Initial DNA strand First PCR cycle Second PCR cycle Third PCR cycle Fourth PCR cycle How to measure the PCR product?

26. Any increase in fluorescence level can be plotted onto a graph and easily interpreted Virus present No virus

27. Denaturation Extension Quantitate Melting curve Annealing

28. We describe the position of the lines with a value that represents the cycle number where the trace crosses a threshold. This is called the cycle threshold “Ct Value”. Ct values are directly related to the starting quantity of DNA, by way of the formula: 23 25 28 Ct Values:

29. Quantitative PCR – in depth Major assay types Hydrolysis probes Basis of TaqMan ® chemistry Uses two primers and an internal hydrolysis probe Most commonly used for fish health diagnostics SYBR ® green dye Increased fluorescence when bound to dsDNA Slightly lower specificity Costs less May not be as sensitive as the 5’ nuclease assays

30. SYBRGreen

31. PCR makes more double-stranded DNA SYBR Green dye binds to dsDNA When illuminated with light at 490nm, the SYBR+DNA complex fluoresces at 520nm. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ Taq Taq 3’ 5’ 3’ Taq Taq 5’ 5’ l l l l l

32. Denaturation Extension Quantitate Melting curve Annealing

33. 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ ID ID ID 5’ 5’ 3’ 3’ ID Melt curves can tell us what products are in a reaction. PCR products that are shorter will melt at lower temperatures. Different PCR products will therefore have different shaped curves.