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SOUTHERN BLOTTING

SOUTHERN BLOTTING. OUTLINE. DNA SPECIMEN COLLECTION AND STORAGE PROCEDURE WATCHPOINTS USES. DNA. Each individuals unique genetic blueprint is stored in material known as DNA. DNA is found in all cells containing a nucleus.

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SOUTHERN BLOTTING

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  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

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