Técnica molecular RFLP PCR (polimorfismo de longitud de fragmentos de restricción)

técnica de fragmentos de restricción polimorfismo

1. RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) Presented by: Muhammad faiz

2. Alec Jeffreys in 1984 Royal Society’s Royal Medal in 1994 Wolf Prize in Medicine in 1998 Discovery

3. Length of DNA Fragments generated by restriction enzyme vary from one plant variety to other Variation due to VNTRs Genetic variation among individuals or population

4. We have 3 Reference DNA samples from 3 Different Varieties & 4 th Sample used to compare with other samples. We have 2 restriction endonuclease enzyme for double digestion To perform RFLP technique

5. DNA Extraction

6. DNA Sample = 15 microliter 10X Assay Buffer = 3.0 microliter Molecular Biology Grade Water = 10 microliter ECoR1 = 1.0 microliter Pst1 = 1.0 microliter Reaction Mixture for RFLP

7. Loading 15 micro litre DNA Sample in microfuge tube

8. Loading 3 micro litre 10X Assay buffer in microfuge tube

9. Adding 10 micro litre Molecular Grade Water in microfuge tube

10. ECOR1 Restriction Endonuclease Enzyme Restriction Digest

11. Loading 1 micro litre ECOR1 in microfuge tube

12. Restriction Digest Pst1 Restriction Endonuclease Enzyme

13. Loading 1 micro litre Pst1 in microfuge tube

14. Incubation at 37 degree for 2-3 hours

15. Adding 6X Gel loading dye to all 4 samples

16. Purpose of loading DNA Ladder DNA size marker or molecular weight marker A mixture of DNA fragments of known sizes To compare size of DNA fragments of experimental sample

17. Loading 1kb or 3 microliter DNA Ladder in first Well

18. Loading 25 micro litre DNA solution mixture to wells

19. 70 Volts for about 45 Minutes

20. Denaturation Gel placed in Sodium hydroxide (NaOH) for Denaturation Single Stranded DNA formed Heating the DNA fragments in an alkaline solution(NaOH ) which break hydrogen bonds

21. Blotting DNA fragments tranfer onto nitrocellulose or nylon membrane. To Immobilize the DNA To increase Sensitivity To Probe the DNA Identify presence of gene of interest from complex mixture Purpose

22. 1_Place Sponge blotting paper after dipping with tranfer buffer 2_Another Sponge paper after dipping with tranfer buffer

23. 3_Place Filter paper after dipping with tranfer buffer 4_Place the Gel on filter paper accurately

24. 5_Place Nylon membrane om Gel 6_Place filter paper on nylon membrane

25. 7_ Place Sponge filter paper on it 8_ Place another Sponge filter paper on it

26. 9_Finally place postive Electrode, a anode on it

27. 10_Place in the tank apparatus 11_Add transfer buffer to tank apparatus

28. Electro plotting transfer apparatus connect with powerpad for 2 hours

29. Cross linking of DNA onto Nylon membrane under UV Light for 20 Minutes

30. Incubation at 70 degree for 20 minutes

31. Add 10ml pre hybridization buffer Incubate at 70rpm for 45 Minutes

32. Add 10 ml of Hybridization buffer at Room Temperature

33. Dip Probe in boiling water bath for 5 min Biotinylated probe, a short sequence of nucleotides complementary to gene of interest

34. Place Probes on ice for 5 min Approximately 15 micro litre probe added in solution on petri dish

35. Add 10ml wash buffer 1 Incubate for 5 min And repeat 3 times

36. Add Blocking buffer 10ml for 1 hour at Room Temperature

37. Add 9ml of conjugate dilution buffer in flask Add 9 microliter of Toeen 20 in same flask

38. Add 6 microliter of streptavidine-HRP &place 20 minutes at Room Temperature Add 10 ml wash buffer III at room temperature, 5 min

39. Add wash buffer 4 Shake until blue color produce

40. Now observation of hybridized probe with gene of interest

41. Gene of interest is detected finally

42. APPLICATIONS 1_ Genetic Mapping 2_ DNA Finger Printing (In forensic, paternity testing, criminal investigations) 3_ Disease Diagnosis 4_ Evolutionary Studies

43. Criminal Investigation

44. Paternity testing

45. Disease Diagnosis

46. Advantages 1_ High Resolution 2_ Wide applicability 3_ Stable 4_ Co dominant

47. 1_ Time Consuming 2_ Low throughput 3_ Radioactive Labelling 4_ Limited markers 5_Interpretation Disadvantages

48. Jazakallah