COMPUTER EXERCISE Design of PCR and PCR-RFLP experiments

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. Toperform a PCR-RFLP experiment, weneed a DNA sample.

3. Grow the problem cells

4. Pick up some bacteria

5. Re-suspend the cells in the suitable buffer

6. Re-suspend the cells in the suitable buffer

7. Apply the desired DNA extraction procedure

8. Thepurified DNA istheproblemsample

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

10. MgCl2 Primers Sample Buffer Taqpol. H2O dNTP The reagents for the PCR reaction must be mixed in a new tube

11. MgCl2 Primers Sample Buffer Taqpol. H2O dNTP dd H2O Add the sterile double-deionized water

12. MgCl2 Primers Sample Buffer Taqpol. H2O dNTP Buffer Add the 10X PCR buffer

13. MgCl2 Primers Sample Buffer Taqpol. H2O dNTP Mg Mg MgCl2 Add the MgCl2+ Magnesium ion serves as cofactor for Taq polymerase.

14. MgCl2 Primers Sample Buffer Taqpol. H2O dNTP dNTP Add the dNTPs mix (dATP, dTTP, dGTP and dCTP)

15. MgCl2 Primers Sample Buffer Taqpol. H2O dNTP Primers Add the primers (forward and reverse primers)

16. Taqpol. MgCl2 Primers Sample Buffer Taqpol. H2O dNTP Add the Taq DNA polimerase

17. MgCl2 Primers Sample Buffer Taqpol. H2O dNTP Sample Add the sample (template DNA)

18. Mg Mg MgCl2 Taqpol. 3´ 5´ 5´ 3´ Sample G C A Primers T dNTP The tube will contain all reagents required for PCR reaction

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

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

21. Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C Primer 1 recognitionsite 5´ 3´ 3´ 5´ Primer 2 recognitionsite Firstcycle, annealingstep: Forward and reverse primerswillbindtotheir target sequences.

22. Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´ 3´ 3´ 5´ Firstcycle, extensionstep: Polymerization of DNA byTaqpolymerase

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

24. Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´ 3´ 5´ 3´ 5´ 3´ 3´ 5´ Firstcycle, extensionstep: Polymerization of DNA byTaqpolymerase

25. Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ Secondcycle, denaturationstep

26. Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ Secondcycle, annealingstep

27. Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ Secondcycle, annealingstep

28. Denaturation: ~ 1 min 90°C Annealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C 5´ 3´ 5´ 3´ 5´ 3´ 3´ 5´ 5´ 5´ 3´ 5´ 3´ 3´ 3´ 5´ Secondcycle, extensionstep

29. 4rd cicle 3rd cicle 2nd cicle 1st cicle n cycles Original DNA 2n copies 24 copies 23 copies 22 copies 21 copies In each cycle, the number of target DNA copies will double

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. Enzyme Sample Buffer H2O Fordigestion of thesample DNA (amplicons) thereagentsmust be mixed in a new tube

33. Enzyme Sample Buffer H2O ddH2O Addthesterile doble-deionizedwater

34. Enzyme Sample Buffer H2O Buffer Addthe 10X buffer

35. Enzyme Sample Buffer H2O Enzyme Add the restriction endonuclease

36. Enzyme Sample Buffer H2O Sample Addthesample DNA (thepurifiedamplicons)

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

38. T T G A A C G G A A T T C C A T G C T A G A T C T C T G C A C G G G A A T C 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 For visualization, the gel will be stained.

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

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

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