Southern, Northern and Western blotting

1. Southern, Northern and Western blotting

2. Comparison of Southern, Northern, and Western analyses of Gene X

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 hybridization Transfer buffer

19. Detection of an RFLP by Southern blotting

20. Detection of the sickle-cell globin gene by Southern blotting

21. Checking of the gene knockout mice

22. Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection Isotope Non-isotope

23. Preparing the samples and running the gel • Digest 10 pg to 10 g of desired DNA samples to completion. • Prepare an agarose gel, load samples (remember marker), and electrophorese. • Stain gel ethidium bromide solution (0.5 g/ml). • Photograph gel (with ruler).

24. Critical parameters (I) • Note the complexity of DNA • Genomic DNA • A single-copy of mammalian gene, 3 Kb average in length 10 mg x 3 Kb/3 x 106 Kb = 10 mg x 1/106 = 10 pg • Plasmid DNA or PCR products 0.1 mg of a 3 Kb plasmid DNA 100 ng

25. Gel treatment • Acid treatment • 0.2 N HCl solution • Denaturation • NaOH solution • Neutralization • Tris-Cl buffer (pH8.0)

26. Southern transfer • Measure gel and set up transfer assembly: • Wick in tray with 20x SSC • Gel • Nitrocellulose or Nylon filters (soaked in H2O and 20x SSC) • 3MM Whatman filter paper • Paper towels • Weight

27. After Southern transfer • Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC • Bake nitrocellulose at 80C for 2 hr or UV-crosslink Nylon membrane for seconds

28. Preparation of probes • Synthesis of uniformly labeled double-stranded DNA probes • Preparation of single-stranded probes • Labeling the 5 and 3 termini of DNA

29. Synthesis of double-stranded DNA probes • Nick translation of DNA • Labeled DNA probes using random oligonucleotide primers

30. Nick translation

31. Preparation of single-stranded probes • Synthesis of single-stranded DNA probes using bacteriophage M13 vectors. • Synthesis of RNA probes by in vitro transcription by bacteriophage DNA-dependent RNA polymerase.

32. In vitro transcription

33. Labeling the 3 termini of double-stranded DNA using the Klenow fragment of E. coli DNA polymerase I. (lack of 5’ 3’ exonuclease activity) Labeling the 3 termini of double-stranded DNA using bacteriophage T4 DNA polymerase. Labeling the 5 termini of DNA with bacteriophage T4 polynucleotide kinase. Labeling the 5 and 3 termini of DNA

34. T4 polynucleotide kinase activity

35. Non-isotope labeling • Digoxigenin-11-dUTP (DIG-dUTP) labeling • DNA labeling • Oligonucleotide labeling • RNA labeling

36. PCR Labeling, Random Primed Labeling, and RNA Labeling

37. Prehybridization • Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr

38. Hybridization • Remove prehybridization solution and add hybridization solution • Add 500,000 cpm of the probe/ml hybridization solution. • Hybridize overnight at appropriate temperature.

39. Post-hybridization washing • Wash twice, 15 min each, in 1x SSC, 0.1% SDS at room temperature. • Wash twice, 15 min each, in 0.25x SSC, 0.1%SDS at hybridization temp

40. Critical parameters (II) • Homology between the probe and the sequences being detected • Tm = 81 +16.6 (log Ci) + 0.4 [% (G+C)] - 0.6 (% formamide)- 600/n - 1.5 (% mismatch) • Factors can be changed: • Hybridization temp. • Washing temp. • Salt concentration during washing High temp., low salt: high stringency Low temp., high salt: low stringency • If 50 % formamide is used • 42 oC for 95 ~ 100 % homology • 37 oC for 90 ~ 95 % homology • 32 oC for 85 ~ 90 % homology

41. Comparison of nitrocellulose and nylon membranes

42. Signals detection • Autoradioragraphy • Non-isotope detection system • Chemiluminescent detection • Colorimetric detection • Multicolor detection

43. Autoradiography • Exposure to x-ray film

44. 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.

45. 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.

46. SOUTHERN BLOTTING

47. 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.

48. 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.

49. 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.

50. 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.