Presented by: Group 8 @ 23 March 2009

1. MB 206 Microbial Biotechnology Presentation Southern, Northern, western Blot Presented by: Group 8 @ 23 March 2009

2. OBJECTIVES * To understand the techniques of molecular searching ( Western, Northern, Southern Blots) * To differentiate the advantages and disadvantages of the techniques * To determine the applications of the techniques used

3. INTRODUCTION

4. Southern, Northern and Western Blot: • Techniques of Molecular Searching • - determine by analyzing cellular molecules (DNA, RNA • and protein) • - transfer the cellular molecules onto a carrier (membrane) • - i.e. after the gel electrophoresis - transfer the molecules • from the gel to the blotting membrane - the transferred • cellular molecules can be detected

5. It can be used as analytical tools based on; Complementarity and Hybridization

6. Southern, Northern and Western Blot

7. Southern Blots • invented by the English Molecular BiologistEdwin Southern (1975) • determine the presence of a specific DNA sequence within a large, complex DNA sample • Probe with radioactive DNA • DNA cut with restriction enzymes is separated by molecular weight • Identify which DNA fragments obtained from a digest of a larger DNA clone that contains sequence complementary to a specific probe • Determine the number of copies of a particular DNA sequence presented in the genome • Can identify related sequences in the genome

9. Northern Blot • Developed by James Alwine, David Kemp, and George • Stark (1977) • Similar to Southern Blotting • Detects the presence a specific mRNA in a total RNA extract • Can determine whether the gene is transcribed or not • Identify where and when it is transcribed • Probed with radioactive DNA or RNA • RNA denatured with formaldehyde (separated by molecular weight)

11. Western Blots • Developed by W. Neal Burnette (1981) • Also known as immunoblot • Detects the presence of specific proteins in a given sample of protein extract • The procedures are rather similar to Southern and Northern except that the cellular content extracted is protein • Protein probed with radioactive or enzymatically-tagged antibodies • Gives information on the size of proteins and expression amounts of the protein • Based on protein-protein interaction; Enzyme Link Immunosorbant Assay (Elisa) • Protein denatured with SDS (separated by molecular weight)

12. Courtesy of www.molecularstation.com

13. METHODOLOGY SOUTHERN BLOTTING NORTHERN BLOTTING WESTERN BLOTTING

14. 3 Blotting. Transfer separated DNA onto membrane for further analysis 1 2 4 6 Digest DNA with restriction enzymes Restriction Fragments are separated by size by agarose gel Synthesis of labelled probe Wash the filter and expose the film to x-ray 5 Hybridize the target DNA with specific labeled probe

15. Southern Blotting Steps: • DNA Fragmentation • Agarose Gel Electrophoresis • Depurination (optional) • Neutralization • Blotting • Prehybridization and Hybridization • Removal of Unbound Probe • Autoradiograph

16. 1. DNA Fragmentation • DNA is digested by one or several restriction enzymes or restriction endonucleases - bacterial enzymes - cut at specific sequence (restriction site) • If REs with different buffer requirements are used, a prior addition of RE buffer before the second enzyme is used.

17. 2. Agarose Gel Electrophoresis • Restriction fragments are separated electrophoretically by size on agarose gel. - negatively charged • DNA fragments migrate into gel toward the anode (+ve electrode) under the influence of electric field.

18. Rate of movement is determined by size of fragment • the largest • molecule • has the lowest • mobilities

19. 3. Depurination • Optional • Occurs before neutralization • When DNA fragments is > 15 kilobases, it is too hard to be transferred to filter • The gel is treated with dilute acid (0.2 M HCl for 15 minutes) • depurinate DNA fragment into smaller pieces and promote higher efficiency transfer to filter.

20. Only ssDNA can be transferred to filter 4. Neutralization • DNA is placed into an alkaline solution containing 0.5mM NaOH to denature the dsDNA into ssDNA and neutralize the acid in previous step. • Function: (1) improve binding of the –vely charged DNA to +vely charged filter (2) ssDNA strands for hybridization (3) destroy any remaining RNA present in the sample

21. Transfer is done by capillary action which draws buffer (binds ssDNA) up onto the membrane The binding of DNA to membrane is due to ion exchange interactions 5. Blotting Exert pressure evenly to a gel to ensure even contact between gel and membrane

22. 5. Blotting (continued) • To permanently attach the transferred DNA to the membrane, the blot can be: -baked in a vacuum or regular oven at 80 °C for 2 hours - exposed to UV radiation

23. 6 (a). Prehybrization • Prevent the labeled probe from binding nonspecifically to DNA fragments on the membrane • Non-specific ssDNA is added such as salmon or herring sperm DNA; deionized formamide, and detergents such as SDS to reduce non-specific binding of the probe

24. Probe • It can be a purified RNA, a cloned cDNA, or a short synthetic oligonucleotide with a reporter substance attached to it. - is a radioactive element like (32P) that induces light production • It contains a short segment of ssDNA that is complementary to the DNA sequence of interest and tags the sequence of interest. • Usually prepared by making a radioactive copy of a DNA fragment. - E.g 32P-labeled probe

25. 6 (b) Preparation of Labeled-probe Treat with DNase (causes double stranded nick in DNA) Add 32P, dATP, and other dNTPs to DNA polymerase I 32P becomes incorporated into, and thus labels, the DNA Heat and on ice to prevent two strands from reannealing

27. 6 (c). Hybridization • Use the same buffer as for prehybridization • The filter is removed and hybridized with a radioactively labeled probe at 65oCand incubated for several hours to allow the probe molecules to find their targets.

28. 7. Removal of Unbound Probe • Unbound probeis washed off and the membrane is exposed to x-ray film.

29. 8.Autoradiograph • The location of the probe is revealed by converting a colorless substrate to a colored product that can be seen or gives off light which will expose X-ray film. • If you used a radiolabeled • 32 P probe, then you would visualize by autoradiograph. • The bands indicate the number and size of the DNA fragments complementary to the probe.

30. Comparison of Nitrocellulose, Nylon membranes

31. Northern Blotting

32. Steps in Northern Blotting: • Extraction of RNA - The RNA sample can be: i. total RNA isolated from particular samples ii. RNA containing poly(A) tails, i.e messenger RNA(mRNA)

33. 2. Gel Electrophoresis - agarose gel 3. The RNA molecules in the gel are transferred to nitrocellulose or nylon. The principle and procedure for Northern Blotting is similar, except you are working with RNA instead of DNA.

34. 1 Gel electrophoresis 3 Labeling with primary antibody Electroblotting 2 Labeling with 2nd antibody 4 Visualization 5 Western Blotting Blocking step

35. 1. Gel electrophoresis

36. 2. Electroblotting • uses an electric current to drive the protein (polypeptide) bands onto the nitrocellulose membrane • It is often be used with gels made of polyacrylamide rather than that of agarose since polyacrylamide has a higher melting temperature. • Protein binding is based upon hydrophobic interactions, as well as charged interactions between the filter and protein.

37. Blocking Step • The nitrocellulose is then soaked into a concentrated nonantigenic protein solution (blocking solution containing nonfat dried milk [BLOTTO]) • The protein in the solution will bind nonspecifically to all areas on nitrocellulose that do not absorb protein from the SDS-polyacrylamide gel - The antibodies are diluted in this nonantigenic protein solution before applying to the nitrocellulose

38. Blocking Step (Continued) • Functions: - prevent the antibodies from binding non-specifically to the nitrocellulose and unrelated proteins on nitrocellulose - increase the probability that they bind to immobilized antigenic proteins

39. 3. Labeling with Primary Antibody • forms an antibody-protein complex with the protein of interest

40. 4. Labeling with Secondary Antibody • Is conjugated to HRP (horseradish peroxidase) • Acts as antibody against primary antibody • Antigens can be visualized through coloured reaction • Advantage: signal of both minor and major antigens can be visualized and optimized on single blot by varying the exposure time

41. 5. Visualization • The position of protein of interest is marked by visible band, forming protein-primary antibody-secondary antibody-enzyme complex • A flash light is observed which expose x-ray film. The light is due to the release of protons by catalyzing the oxidation luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) by HRP.

42. The advantages and disadvantages of Southern, Northern and Western Blots (Techniques of molecular searching)

43. Gene Cloning • Basic Steps in Gene Cloning: DNA selection Cut DNA and vectors with restriction endonuclease Insert DNA fragments into vectors Seal the vector and DNA fragment with ligase Transferred the recombinant DNA into bacterial cells Plate the cells on agar plate with antibiotics

44. Gene Library • Cloned genes with different DNA fragments from 1 organism Gene library of the organism • Gene library  used to screen for gene of interest

45. Gene Library • Different types of gene library

46. Screening of Gene Library • Uses: • To identify the gene of interest • Techniques: • Southern blotting • Northern blotting • Western blotting

47. Screening of Gene Library • Southern blotting ~ Detect gene fragments of interest 2. Northern blotting ~ Detecttranscription (mRNA) level 3. Western blotting ~ Detect the fusion protein (target protein) with a specific antibody ~ Mostly used in libraries in phage λ expression vectors

48. Southern Blotting • Main functions • Detect the specific DNA sequence (gene) of interest • Determine the length of the restriction fragment carrying the sequence • Detect the restriction site

49. Southern Blotting • Application • Diagnosis of human disease • Detect point mutation, gene rearrangement or gene amplification • Mutated gene  change in the size (hemophilia A) • Gene rearrangement  change in size and pattern (leukemia) • Amplification  increase in gene copy number (Charcot-Marie-Tooth syndrome)

50. Southern Blotting • Application (continue) • Identify structurally related genes in the same species or among other species • Understand various biological processes • Discovery of RNA splicing, genomic rearrangement to form antibodies and T cell receptors and etc. • Construct a restriction map of a specific gene • By performing RE digestion to the specific gene