Blotting - Northern, Southern, Western

This PPT discusses about the main types of Nucleic Acid Based Techniques - Blotting (Southern, Northen, Western)

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1. Nucleic Acid Based Techniques - Blotting Presented By: Nizam Ashraf IInd SEM M.Sc. Marine Microbiology OST-2018-22-15

2. Blotting is technique in which nucleic acids i.e., DNA/RNA/Proteins are transferred onto a specific membrane. This membrane may be nitrocellulose PVDF ( Polyvinylidene fluoride) or nylon membrane. The blotting process is carried just after the gel electrophoresis, by transferring the molecules from the gel onto the surface of blotting membrane. Later, the molecules are visualized by staining. Examples : Ethidium bromide ( EtBr ) , Crystal violet, Saffranine and Ossmium tetroxide etc

3. Types of Blotting SOUTHERN BLOTTING NORTHERN BLOTTING WESTERN BLOTTING

4. It was developed by Edward M. Southern (1975). Southern blotting is a hybridization technique for identification of particular size of  DNA  from a sample. This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labelled probe hybridization. Basically, the DNA fragments are separated on the basis of size and charge during electrophoresis. Separated DNA fragments after transferring on nylon membrane, the desired DNA is detected using specific DNA probe that is complementary to the desired DNA. A hybridization probe is a short (100-500bp), single stranded DNA. The probes are labeled with a marker so that they can be detected after hybridization. SOUTHERN BLOTTING

5. Principle The key to this method is hybridization. Hybridization: It is the process of forming a double stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA. The important features of hybridization: The reactions are specific : the probes will only bind to targets with a complementary sequence.

6. ( Agarose ) Filter Paper Bridge Buffer

7. Procedure

8. Advantages / Applications DNA finger printing : P aternity testing criminal identification victim identification To isolate and identify desire gene of interest. Used in RFLP To identify mutation or gene rearrangement in the sequence of DNA D iagnosis of disease caused by genetic defects Used to identify infectious agents

9. Disadvantages More expensive than most other tests. Complex and labor-intensive. Time consuming.

10. The northern blot is a technique used in molecular biology to study gene expression by detection of RNA (or isolated mRNA) in a sample. Developed by Alwnie and his colleagues in 1979. This method was named for its similarity to the technique known as a Southern blot NORTHERN BLOTTING

11. Procedure Extract and purify mRNA from the cells. Separate these mRNA on agarose gels containing formaldehyde as a denaturing agent for the RNA This gel is immersed in depurination buffer for 5-10 minutes and washed with water Then transfer these mRNA fragments onto the carrier membrane i.e., aminobenzyloxymethyl (ABOM) filter paper After transferring the mRNA, it is fixed to the membrane by using UV or heat . (A process known as Baking, which is carried out at 80C). Add DNA labeled probe for hybridization. Wash off the unbound probe and at the end mRNA-DNA hybrid are then detected by X-ray film using Autoradiography

12. Advantages A standard for direct study of the gene expression at the level of mRNA. Detection of mRNA transcript size Time consuming procedure . Use of radioactive probes. Detection with multiple probes is a problem. Disadvantages

13. WESTERN BLOTTING Western blotting is an immunoblotting technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a sample. In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein. A primary antibody direct against protein of interest and then a secondary antibody is used to detect the primary antibody and gives either colored or chemiluminescent signal. The SDS PAGE technique is a prerequisite for Western blotting .

14. Procedure Firstly , isolate the protein from particular sample. After that beta- mercaptoethanol (BME) and Sodium dodecyl Sulfate (SDS) is added into the protein suspension. Then , protein- SDS complex is placed on top of the gel in the well . Once the samples and markers are loaded then current is passed across the gel. Protein is pulled down to the positive pole of the well because it is tightly bound to SDS which is negatively charged. Movement of protein is inversely proportional to its size After this step, gel is placed against a membrane and current is passed across the gel so that all the proteins are transferred onto the membrane

15. Then i mmunoblotting has to be done. In this method, firstly block the membrane with non-specific protein in order to prevent antibody from binding to the membrane where the protein is not present. After that primary antibody is added to the solution. These antibodies are responsible for recognizing a specific amino-acid sequence. Then wash it to remove unbound primary antibody and add secondary antibody. Now these antibodies are conjugated with an enzyme and recognize the primary antibody. Lastly, another wash is done to remove unbound secondary antibody. Here, chemiluminescent substrate is used for detection. The light is being emitted once the substrate has been added and can be detected with film imager.

16. Application The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Western blot can also be used as a confirmatory test for Hepatitis B infection. If a protein is degraded quickly, Western blotting won't detect it well. This test takes longer that other existing tests It might also be more costly Disadvantages

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