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Welcome

Welcome. Polymerase Chain Reaction PCR. Subjects to be discussed. Definition Targets of PCR Requirements of PCR Denaturing of DNA PCR Primers Primer annealing PCR DNA Taq polymerase PCR Cycles Application of PCR . DNA Molecule. Adenine Thymine Guanine Cytosine.

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  1. Welcome

  2. Polymerase Chain Reaction PCR

  3. Subjects to be discussed • Definition • Targets of PCR • Requirements of PCR • Denaturing of DNA • PCR Primers • Primer annealing • PCR DNA Taq polymerase • PCR Cycles • Application of PCR

  4. DNA Molecule Adenine Thymine Guanine Cytosine

  5. Polymerase Chain Reaction (PCR)Definition PCR (Polymerase Chain Reaction) A rapid and simple method for amplification of very small amounts of DNA into multiple copies Which are used for further testing. e.g. diagnosis of genetic disease

  6. PCR Target The targets in PCR are: sequences of DNA which either a complete gene or small sequence.

  7. PCR Targets The number of bases in the targets can vary. TTAAGGCTCGA . . . . AATTGGTTAA The . . . . Represents the middle DNA sequence, and does not have to be known to replicate it.

  8. PCR Requirements • Magnesium chloride: 0.5-2.5 mM • Buffer: pH 8.3-8.8 • dNTPs: 20-200µM • Primers: 0.1-0.5µM • DNA Polymerase: 1-2.5 units • Target DNA:  1 µg

  9. PCR Denaturing Denaturing is the first step in PCR, in which the DNA strands are separated by heating to 95°C.

  10. PCR Primers Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the complementary building blocks of the target sequence.

  11. PCR Primers A primer for each target sequence on the end of your DNA is needed. This allows both strands to be copied simultaneously in both directions.

  12. PCR Primers TTAACGGCCTTAA . . . TTTAAACCGGTT AATTGCCGGAATT . . . . . . . . . .> And <. . . . . . . . . . AAATTTGGCCAA TTAACGGCCTTAA . . . TTTAAACCGGTT

  13. PCR Primers The primers are added in excess so they will bind to the target DNA instead of the two strands binding back to each other.

  14. PCR Annealing Annealing is the process of allowing two sequences of DNA to form hydrogen bonds. The annealing of the target sequences and primers is done by cooling the DNA to 55°C.

  15. PCR Taq DNA Polymerase Taq stands for Thermus aquaticus, which is a microbe found in 176°F hot springs in Yellow Stone National Forest.

  16. PCR Taq DNA Polymerase Taq produces an enzyme called DNA polymerase, that amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg.

  17. PCR Cycles

  18. In PCR, the template DNA to be amplified is first heated to 90-95 °C, to cause strand separation. • Then cooled to 50-65 ° C in the presence of the two primers to allow annealing of the primers to their respective DNA sequences.

  19. The reaction temperature is then adjusted to 72oC in the presence of heat-stable Taq polymerase. • The Taq polymerase leads to a primer-mediated extension of both strands of the template DNA, in the region between the two primers.

  20. The three reactions : 1. Denaturation 2. Primer-annealing 2. Primer-directed extension Those reactions are repeated, leading to a geometric increase in the DNA between the two primers

  21. dNTPs Primers DNA polymerase

  22. dNTPs Primers DNA polymerase

  23. dNTPs Primers DNA polymerase

  24. Denature at 95ºC dNTPs Primers DNA polymerase

  25. Denature at 95ºC dNTPs Primers DNA polymerase

  26. Denature at 95ºC dNTPs Primers DNA polymerase

  27. Anneal primers dNTPs Primers DNA polymerase

  28. Anneal primers dNTPs Primers DNA polymerase

  29. Anneal primers dNTPs Primers DNA polymerase

  30. Anneal primers dNTPs Primers DNA polymerase

  31. Extend at 72°C dNTPs Primers DNA polymerase

  32. Extend at 72°C dNTPs Primers DNA polymerase

  33. Extend at 72°C dNTPs Primers DNA polymerase

  34. Extend at 72°C dNTPs Primers DNA polymerase

  35. Extend at 72°C dNTPs Primers DNA polymerase

  36. Extend at 72°C DNA polymerase Primers dNTPs

  37. Extend at 72°C DNA polymerase Primers dNTPs

  38. Extend at 72°C DNA polymerase Primers dNTPs

  39. DNA polymerase Primers dNTPs

  40. DNA polymerase Primers dNTPs

  41. DNA polymerase Primers dNTPs

  42. Denature at 95ºC DNA polymerase Primers dNTPs

  43. Denature at 95ºC DNA polymerase Primers dNTPs

  44. Anneal primers DNA polymerase Primers dNTPs

  45. Extend at 72°C Primers dNTPs

  46. Extend at 72°C Primers dNTPs

  47. Extend at 72°C Primers dNTPs

  48. Extend at 72°C Primers dNTPs

  49. After several cycles

  50. The theoretical amplification achieved is 2 n, where (n) is the number of cycles of denaturation-annealing-extension. • Using this reaction, specific lengths of DNA can be amplified a million-fold, to an extent that the amplified product can be directly visualized on an agarose gel without the need for a Southern blot to be carried out using a labelled probe.

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