1. COMPUTER EXERCISE Design of PCR and PCR-RFLP experiments This presentation shows all steps of a PCR-RFLP experiment and is a companion of the computer exercise at http://insilico.ehu.es/edu

2. To perform a PCR-RFLP experiment, we need a DNA sample.

3. Grow the problem cells

4. Pick up some bacteria

5. Re-suspend the cells in the suitable buffer

7. Apply the desired DNA extraction procedure

8. The purified DNA is the problem sample

9. In this presentation, we will consider two samples obtained from two different bacterial strains

10. The reagents for the PCR reaction must be mixed in a new tube H2OH2O Buffer MgCl 2 dNTP Primers Taq pol. Sample

11. Add the sterile double-deionized water dd H 2 O H2OH2O Buffer MgCl 2 dNTP Primers Taq pol. Sample

12. Buffer Add the 10X PCR buffer H2OH2O Buffer MgCl 2 dNTP Primers Taq pol. Sample

13. Add the MgCl 2+ Magnesium ion serves as cofactor for Taq polymerase. MgCl 2 Mg H2OH2O Buffer MgCl 2 dNTP Primers Taq pol. Sample

14. dNTP Add the dNTPs mix (dATP, dTTP, dGTP and dCTP) H2OH2O Buffer MgCl 2 dNTP Primers Taq pol. Sample

15. Primers Add the primers (forward and reverse primers) H2OH2O Buffer MgCl 2 dNTP Primers Taq pol. Sample

16. Taq pol. Add the Taq DNA polimerase H2OH2O Buffer MgCl 2 dNTP Primers Taq pol. Sample

17. Add the sample (template DNA) H2OH2O Buffer MgCl 2 dNTP Primers Taq pol. Sample

18. The tube will contain all reagents required for PCR reaction 5´ 3´ 5´ MgCl 2 Taq pol. Sample Mg dNTP Primers G A C T

19. During PCR reaction the following steps will be repeated 20 to 40 times: denaturation, annealing and extension Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´3´ 3´5´

20. First cycle, denaturation step: The DNA strands are separated Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´3´ 3´5´

21. 5´3´ First cycle, annealing step: Forward and reverse primers will bind to their target sequences. Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C Primer 1 recognition site Primer 2 recognition site 3´5´

22. 5´3´ 3´5´ First cycle, extension step: Polymerization of DNA by Taq polymerase Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C

23. T G C C T A G T A G T C G C Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C C G G A C T G A T C A T A C G T A G C A T A Mg During extension, nucleotides complementary to target sequence are incorporated in the new DNA strand

24. 5´3´ 3´5´ Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´ 3´ First cycle, extension step: Polymerization of DNA by Taq polymerase

25. 5´3´ 3´5´ 5´3´ 3´5´ Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C Second cycle, denaturation step

26. 3´5´ 5´3´ 3´5´ Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C Second cycle, annealing step

27. 5´3´ 3´5´ Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 3´5´ 5´3´ Second cycle, annealing step

28. 5´3´ 3´5´ Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 3´ 5´ 3´5´ 5´3´ 3´ Second cycle, extension step

29. In each cycle, the number of target DNA copies will double 1st cicle 2nd cicle 3rd cicle 4rd cicle n cycles 2 1 copies 2 2 copies 2 3 copies 2 4 copies 2 n copies Original DNA

30. Prior to RFLP, it is convenient to purify the DNA sample to avoid inhibition of the restriction endonuclease activity

31. Many copies of the purified amplicons will be obtained. They will be the sample for therestriction step

32. For digestion of the sample DNA (amplicons) the reagents must be mixed in a new tube H2OH2O Buffer Enzyme Sample

33. Add the sterile doble-deionized water ddH 2 O H2OH2O Buffer Enzyme Sample

34. Buffer Add the 10X buffer H2OH2O Buffer Enzyme Sample

35. Add the restriction endonuclease Enzyme H2OH2O Buffer Enzyme Sample

36. Add the sample DNA (the purified amplicons) H2OH2O Buffer Enzyme Sample

37. C C G T A C G C G T A T A G C G A T A T C G T A T A C G T A C G T A C G T A C C G T A C G C G T A T A G C T A T C G T A T A C G T A C G T A C G T A G A During incubation, the restriction endonuclease will specifically recognize the target sequence.

38. C C G T A C G C G T A T A G C G A T A T C G T A T A C G T A C G T A C G T A And it will be cleaved.

39. For visualization of the PCR-RFLP experiment DNA samples will be electrophoretically separated in an agarose gel.

40. Ladder Molecular weight standards will be added to one line.

41. Sample A In the second line, sample A will be added. In this example, amplicons in this sample were not cleaved by the endonuclease.

42. Sample B In the third line, sample B will be added. In this example, amplicons were cleaved by the endonuclease.

43. During electrophoresis, the ladder and de samples will migrate within the agarose gel.

44. LAB LAB For visualization, the gel will be stained.

45. 1000 500 400 300 200 100 50 Ladder (pb) LAB LAB The molecular weight standards will be used to compute the weight of the bands in samples A and B.

46. LAB LAB Sample A contains a unique band of approximately 300 bp. This band was not cleaved by the endonuclease.

47. LAB LAB Sample B contains two bands. Cleavage of a 300 bp band containing the target for the endonuclease yielded these 100 and 200 bp bands

48. LAB LAB The PCR-RFLP experiment was able to discern the two samples due to the presence of a target sequence for the endonuclease in sample B.

49. You may try to solve the online PCR-RFLP exercise available at http://insilico.ehu.es/edu