1. 1 Lecture 11 & 12 Dr. Aparna Islam

2. Blotting 2

3. 3 Gel electrophoresis techniques enables us to separate fragments of DNA in a mixture and by staining we can visualize and detect them. Use of specific labeled probes enables in detection and identifications of fragments of DNA by hybridization procedure. To facilitate this hybridization, the bands are often transferred to a nitrocellulose membrane. This technique resembles blotting. For such blotting, DNA has to be single stranded form. This can be achieved by denaturation of DNA. Gel electrophoresis techniques enables us to separate fragments of DNA in a mixture and by staining we can visualize and detect them. Use of specific labeled probes enables in detection and identifications of fragments of DNA by hybridization procedure. To facilitate this hybridization, the bands are often transferred to a nitrocellulose membrane. This technique resembles blotting. For such blotting, DNA has to be single stranded form. This can be achieved by denaturation of DNA.

4. Blotting All techniques use electrophoresis to separate. Difference in techniques lies in the target Four applications – Western – Southern – Northern – Southwestern 4

5. 5 Southern hybridization

6.  ‘Southern’ hybridization named after Sir Edwin Southern  Developed in 1975  One of the most highly cited scientific publications  Earned Sir Southern a Lasker Award in 2005 6 Sir Edwin Southern

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8. 8 In Southern blotting, a DNA fragment containing a specific sequence can be identified by electrophoresis, transferring them into nitrocellulose & hybridizing with a 32 P labeled single stranded DNA probe complementary to the sequence. The fragment containing the sequence then visualized by audioradiography.

9. Probe A probe is a small (25-2000 bp) length of DNA or RNA – Complementary to the sequence (gene) of interest – Labeled for subsequent detection procedures How do we detect the probe? – P 32, Digoxigenin (DIG) 9

10. PROCEDURE For Southern blotting, DNA sample is first digested with a restriction enzyme and digested sample is electrophoresed. The DNA bands in the gel are denatured into single strands with the help of an alkali solution. Subsequently the gel is laid on top of a buffer saturated filter paper placed on a solid support(eg.glass plate),with its two edges immersed in the buffer. A sheet of nitrocellulose filter membrane is placed on top of the gel and a stack of many papers on top of this membrane. 10

11. Capillary action pulls the buffer from the bottom filter through the gel, to the transfer medium and up through the paper towel stack. While passing through the gel, the buffer carries with its single stranded DNA, which binds on to the nitrocellulose membrane, when the buffer passes through it to the paper towels. The nitro cellulose membrane with single stranded DNA bands blotted on to it, is baked at 80c for 2-3 hours to fix the DNA permanently on the membrane. DNA fragments on membrane can then be probed for sequence of interest by hybridization between them. Membrane is washed to remove the unbound Probe DNA. X-ray film is then exposed to the membrane to get autoradiographs. 11

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15. Step : Detection DIG-labeled probes emitting minute amounts of light (chemiluminescence) 32 P-labeled probes emitting ß-particles 15

16. Detection DIG-labeled probes emitting minute amounts of light (chemiluminescence) 32 P-labeled probes emitting ß-particles Autoradiography film can detect this radiation 16

17. Southern blot analysis:MOM [blue], DAD [yellow], and their four children: D1 (the biological daughter), D2 (step-daughter, child of Mom and her former husband [red]), S1 (biological son), and S2 (adopted son,not biologically related [his parents are light and dark green]). 17

18. Detection of the sickle-cell globin gene by Southern blotting 18

19. APPLICATIONS Southern blotting is extremely sensitive and can be applied to mapping restriction sites around a single copy gene sequence in a complex genome such as that of man. When minisatellite probe is used the technique can be also used for forensic purpose for identification of minute amount of DNA. The use of southern blot technique has also been done for analyzing the role of DNA methylation in gene expression. 19

20. Blotting analysis useful in detection of mutated genes in genetic disorders. Restriction analysis of genes studied with southern blot helps in this respect. RFLP mapping. DNA finger printing. 20

21. 21 Northern blotting

22. 22 The northern blot is used to study the expression patterns of a specific type of RNA molecule as relative comparison among a set of different samples of RNA. RNA is separated based on size and is then transferred to a membrane then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used. Most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. It is used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.

23. Northern Blot Used to study gene expression. Similar to other blots but MAJOR difference is that RNA is analyzed. Gels may be run on either agarose or denaturing polyacrylamide, the latter being preferable for smaller RNA fragments. Formaldahyde is added to gel and acts as a denaturant to agarose. For polyacrylamide, urea is the denaturant. Not used much for diagnostic, mainly used in research. 23

24. Steps in Northern bloting Prepare RNA samples and run RNA gel Northern transfer Probe preparation-isope/nonisotope Prehybridization (WHY?) Hybridization Post-hybridization washing (WHY?) Signal detection 24

25. Northern Blot Three types of RNA: tRNA, rRNA and mRNA Northern blot isolates and hybridizes mRNA Procedure – mRNA extracted from cells and purified – Separate with electrophoresis – Transfer onto membrane – Use labelled probes to identify mRNA of interest 25

26. Northern Blot 26

27. Northern Blot 27

28. APPLICATIONS Used for detection and quantitative estimation of hybridized mRNA. Study RNA degradation Study RNA half life Study RNA splicing It is useful in the studies of gene expression. 28

29. 29 Western Blot

30. A technique used to identify and locate proteins based on their ability to bind to specific antibodies. Detect protein of interest from a mixture of a great number of proteins. Gives information about size of protein in comparison to size marker or ladder. Using western blotting techniques allows not only detection but also quantitative analysis. 30

31. 31 The translated product in the form of proteins can be identified by this technique. In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a nitrocellulose, nylon or other support membrane. This membrane probed with solutions of antibodies. Antibodies specifically bind to the protein of interest & visualized by a variety of techniques, including coloured products, chemiluminescence, or autoradiography. Antibodies are labelled with enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection.

32. Western Blot 32

33. Western Blot Procedure – Separate proteins by SDS-PAGE – Transfer proteins onto membrane – Add primary antibody to protein of interest – Add secondary antibody, specific for primary antibody, attached to an enzyme – Add substrate to visualize bands. Question: What is the purpose of the blocking agent? 33

34. PROCEDURE First we isolate the protein or extract the protein. The extracted proteins are subjected to PAGE(Poly Acrylamide Gel Electrophoresis) and are then transferred onto nitro cellulose to which they bind. Then radiolabelled specific antibody is added on such membrane and it binds only to specific complementary protein. The antibody is labelled and the signal is detected again with autoradiography. If radio active label is not used, bound antibody may be detected by a second antibody tagged with an enzyme. 34

35. Western Blot followed by SDS Proteins are separated using SDS-polyacrylamide gel electrophoresis which separates proteins by size. Nitrocellulose membrane is placed on the gel. The actual blotting process may be active (electroblotting) Electroblotter is used for faster and more efficient transfer of protein from gel to membrane Sandwich of filter paper, gel, membrane and more filter paper is prepared in a cassette An electric current is passed through the gel causing the proteins to electrophorese out of the gel and onto the nitorcellulose membrane. 35

36. Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. This antibody is used as a probe to detect the protein of interest. 36

37. APPLICATIONS 37 1.The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody. 2.A western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease'). 3.Western blot can also be used as a confirmatory test for Hepatitis B infection.

38. 38 Southwestern Blot

39. Combines features of Southern and Western blotting techniques. For rapid characterization of both DNA binding proteins and their specific sites on genomic DNA. Involves identifying and characterizing DNA-binding proteins (proteins that bind to DNA) by their ability to bind to a specific oligonucleotide probes. Identification of protein factors that bind to genes to turn them on or off is therefore important in investigating gene functions. Primary use is for research, not clinical applications. 39

40. Southwestern Blot Procedure – Separate proteins using SDS-PAGE – Renatured by removing SDS in presence of urea – Transfer to membrane – Genomic DNA of interest is digested by restriction enzymes and labeled – Then added to separated proteins. 40

41. Comparison of Southern, Northern, and Western analyses of Gene X 41

42. 42 Southern blottingNorthern blottingWestern blotting Molecule detected DNA (ds)mRNA (ss)Protein Gel electrophoresis Agarose gelFormaldehyde agarose gel Polyacrylamide gel Gel pretreatment Depurination, denaturation, and neutralization -- Blotting methodCapillary transfer Electric transfer ProbesDNA Radioactive or nonradioactive cDNA, cRNA Radioactive or nonradioactive primary antibody Detection system Autoradiography Chemiluminescent Colorimetric Autoradiography Chemiluminescent Colorimetric Chemiluminescent Colorimetric Comparison of Southern, Northern, and Western blotting techniques

43. Comparison of Blotting Methods SouthernNorthernWesternSouthwest ern What is separated DNA cut with restriction enzymes Denatured RNAProtein denatured with SDS Characterizes DNA binding proteins ProbeRadioactive gene X DNA Antibody against protein X, labeled with enzyme or radioactivity Labelled DNA probes What do you learn Restriction map of gene X chromosome How much gene X mRNA is present. How long is gene X mRNA How much protein X is present. How large is protein X. Identify expression of specific DNA binding proteins. 43