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Southern Blotting Technique

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  • 1. SOUTHERN BLOTTING M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.
  • 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, organ s, 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  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.
  • 21. 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.
  • 22. 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.
  • 23. 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.
  • 24. 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.
  • 25. SOUTHERN BLOTTING  This allows for specific sequences to be identified more readily.  Fragments are now easily separated by gel electrophoresis.
  • 26. 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
  • 27. 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.
  • 28. 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.
  • 29. 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.
  • 30. 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.
  • 31. 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.
  • 32. SOUTHERN BLOTTING  DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe.  Transferred by either electrophoresis or capillary blotting.
  • 33. SOUTHERN BLOTTING  1) Electrophoresis- takes advantage of the molecules negative charge.
  • 34. 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.
  • 35. SOUTHERN BLOTTING  The blot is made permanent by: ◦ Drying at ~80°C ◦ Exposing to UV irradiation
  • 36. 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.
  • 37. 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.
  • 38. 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.
  • 39. SOUTHERN BLOTTING◦ 3. DNA polymerase is added with radioactive nucleotides. ◦ 4. The mixture is boiled to separate the strands and is ready for hybridization.
  • 40. 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.
  • 41. SOUTHERN BLOTTING
  • 42. 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.
  • 43. 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.
  • 44. SOUTHERN BLOTTING  IX. Detection ◦ Radioactive probes enable autoradiographic detection.
  • 45. 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.
  • 46. 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
  • 47. 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
  • 48. Watch points  Improper blocking-high background and uninterpretable results.  Insufficient washing-high background and uninterpretable results.  Excess washing- dissociate the specific hybrids.
  • 49. 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
  • 50. USES  Every person has repeated sequences of base pairs which are called Variable Number Tandem Repeats (VNTRs)  To find a particular VNTR we use a radioactive version of the one in question.  This pattern is known as a DNA fingerprint.
  • 51. USES  Applications of DNA fingerprinting include: ◦ Paternity and Maternity Testing ◦ Criminal Identification and Forensics ◦ Personal Identification
  • 52. THANK YOU