SOUTHERN BLOTTING

1. SOUTHERN BLOTTING

2. OUTLINE • DNA • SPECIMEN COLLECTION AND STORAGE • PROCEDURE • WATCHPOINTS • USES

3. DNA • Each individuals unique genetic blueprint is stored in material known as DNA. • DNA is found in all cells containing a nucleus. • DNA can be extracted for analysis from hair, bones, saliva, sperm, skin, organs, all body tissues and blood.

4. DNA • The deoxyribonucleic acid, DNA, is a long chain of nucleotides which consist of: • 1. Deoxyribose(sugar with 5 carbons) • 2. Phosphate groups • 3. Organic(nitrogenous)bases

5. Nitrogenous Bases • Two classes: • Purines • Adenine • Guanine • Pyrimidines • Cytosine • Thymine

6. DNA • DNA molecules are arranged in a double helix which resembles a tightly coiled twisted ladder. • The sides of the ladder have alternating units of phosphate and deoxyribose sugar.

7. DNA • The rungs of the ladder are formed by the nitrogenous “base pairs”. • Hydrogen bonds hold the strands together. • The bases bind together in a complementary fashion.

8. DNA • The base adenine (A) always pairs with thymine (T). • The base guanine (G) always pairs with cytosine (C).

9. DNA • Example • First strand GGGTTTAAACCC • Second strand CCCAAATTTGGG

10. DNA STORAGE AND COLLECTION • I. Temperature Storage for DNA • Purified DNA may be refrigerated at 4°C for up to 3 years. • Samples kept over 3 years should be frozen at -70°C.

11. DNA STORAGE AND COLLECTION • II. Specimens used in DNA testing • Whole blood • Solid tissue • Serum and plasma • Urine • Bone marrow • and many others

12. DNA STORAGE AND COLLECTION • III. Specimen Collection Requirements • A. Blood and Bone Marrow • Collection tubes are EDTA or ACD • 5-15 ml • Samples should not be frozen for transport • 4-25°C

13. DNA STORAGE AND COLLECTION • B. Serum • Collection tubes with no additives • 100 µl to 1 ml • Transported at 20-25°C

14. DNA STORAGE AND COLLECTION • Spin the samples to separate the plasma, RBC, and buffy coat. • Extract the buffy coat • The buffy coat is used because the WBC are nucleated and contain DNA.

15. DNA STORAGE AND COLLECTION • C. Tissue • A sterile container with no formalin or paraffin must be used for collection. • 30 mg • Dry ice should be used for transport.

16. DNA STORAGE AND COLLECTION • D. Urine • Urine container should be used for collection. • At least 1 ml should be collected. • Transported at 4-25°C

17. SOUTHERN BLOTTING • The technique was developed by E.M. Southern in 1975. • The Southern blot is used to detect the presence of a particular piece of DNA in a sample. • The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

18. SOUTHERN BLOTTING • The key to this method is hybridization. • Hybridization-process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

19. SOUTHERN BLOTTING • There are 2 important features of hybridization: • The reactions are specific-the probes will only bind to targets with a complementary sequence. • The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

20. SOUTHERN BLOTTING

21. SOUTHERN BLOTTING • Steps for hybridization • 1. The mixture of molecules is separated. • 2. The molecules are immobilized on a matrix. • 3. The probe is added to the matrix to bind to the molecules. • 4. Any unbound probes are then removed. • 5. The place where the probe is connected corresponds to the location of the immobilized target molecule.

22. SOUTHERN BLOTTING • I. DNA Purification • Isolate the DNA in question from the rest of the cellular material in the nucleus. • Incubate specimen with detergent to promote cell lysis. • Lysis frees cellular proteins and DNA.

23. SOUTHERN BLOTTING • Proteins are enzymatically degraded by incubation with proteinase. • Organic or non-inorganic extraction removes proteins. • DNA is purified from solution by alcohol precipitation. • Visible DNA fibers are removed and suspended in buffer.

24. SOUTHERN BLOTTING • II. DNA Fragmentation • Cut the DNA into different sized pieces. • Use restriction endonucleases (RE) • Bacterial proteins • In vivo, they are involved in DNA metabolism and repair or in bacterial host defense.

25. SOUTHERN BLOTTING • Nucleases hydrolyze the bonds that connect bases within the strand, resulting in cleavage of the strand. • They cleave the double stranded nucleic acid only at specific points.

26. SOUTHERN BLOTTING • This allows for specific sequences to be identified more readily. • Fragments are now easily separated by gel electrophoresis.

27. SOUTHERN BLOTTING • III. Gel Electrophoresis • Sorts the DNA pieces by size • Gels are solid with microscopic pores • Agarose or polyacrimide • Gel is soaked in a buffer which controls the size of the pores • Standards should also be run

28. SOUTHERN BLOTTING • Nucleic acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size.

29. SOUTHERN BLOTTING • Gels can be stained with ethidium bromide. • This causes DNA to fluoresce under UV light which permits photography of the gel. • You can tell the exact migration of DNA standards and the quality of the RE digestion of the test DNA.

30. SOUTHERN BLOTTING • High quality intact DNA should give the appearance of a single band. • Degraded material will smear downwards. • Only a small amount of degradation is tolerable.

31. SOUTHERN BLOTTING • IV. Blotting • Transfer the DNA from the gel to a solid support. • The blot is usually done on a sheet of nitrocellulose paper or nylon.

32. SOUTHERN BLOTTING • DNA is partially depurinated with dilute HCL which promotes higher efficiency transfer by breaking down fragments into smaller pieces. • DNA is then denatured with an alkaline solution such as NAOH. • This causes the double stranded to become single-stranded.

33. SOUTHERN BLOTTING • DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe. • Transferred by either electrophoresis or capillary blotting.

34. SOUTHERN BLOTTING • 1) Electrophoresis- takes advantage of the molecules negative charge.

35. SOUTHERN BLOTTING • 2) Capillary blotting-fragments are eluted from the gel and deposited onto the membrane by buffer that is drawn through the gel by capillary action.

36. SOUTHERN BLOTTING • The blot is made permanent by: • Drying at ~80°C • Exposing to UV irradiation

37. SOUTHERN BLOTTING • V. Blocking • Buffer binds to areas on the blot not occupied by patient DNA. • Blocks the empty sites from being bound during hybridization.

38. SOUTHERN BLOTTING • VI. Preparing the probe • Small piece of DNA used to find another piece of DNA • Must be labeled to be visualized • Usually prepared by making a radioactive copy of a DNA fragment.

39. SOUTHERN BLOTTING • The DNA fragment is labeled by the Random Hexamer Labeling Process: • 1. The template DNA is denatured by boiling. • 2. A mixture of hexamers (6 nucleotides) containing all possible sequences is added and allow to base pair.

40. SOUTHERN BLOTTING • 3. DNA polymerase is added with radioactive nucleotides. • 4. The mixture is boiled to separate the strands and is ready for hybridization.

41. SOUTHERN BLOTTING • The Random Hexamer Labeling Process produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe.

42. SOUTHERN BLOTTING

43. SOUTHERN BLOTTING • VII. Hybridization • The labeled probe is added to the blocked membrane in buffer and incubated for several hours to allow the probe molecules to find their targets.

44. SOUTHERN BLOTTING • VIII. Washing • Excess probe will have bound nonspecifically to the membrane despite the blocking reagents. • Blot is incubated with wash buffers containing NaCl and detergent to wash away excess probe and reduce background.

45. SOUTHERN BLOTTING • IX. Detection • Radioactive probes enable autoradiographic detection.

46. SOUTHERN BLOTTING • If the probe is radioactive, the particles it emits will expose X-ray film. • By pressing the filter and film, the film will become exposed wherever probe is bound to the filter. • After development, there will be dark spots on the film wherever the probe bound.

47. SOUTHERN BLOTTING • Summary of procedure • 1. Extract and purify DNA from cells • 2. DNA is restricted with enzymes • 3. Sort by electrophoresis • 4. Denature DNA • 5. Transfer to nitrocellulose paper • 6. Block with excess DNA • 7. Wash off unbound probe • 8. Autoradiograph

48. Watch points • Using too little DNA-compromise the sensitivity of the test • Using too much DNA- poor restriction enzyme digestion • Using too high voltage setting for electrophoresis- gel to melt or appearance of artifacts

49. Watch points • Improper blocking-high background and uninterpretable results. • Insufficient washing-high background and uninterpretable results. • Excess washing- dissociate the specific hybrids.

50. USES • Identify mutations, deletions, and gene rearrangements • Used in prognosis of cancer and in prenatal diagnosis of genetic diseases • Leukemias • Diagnosis of HIV-1 and infectious disease