Blotting technique including Southern , Northern and Western blotting

he given ppt contains all the blotting techniques which is being studied by students in Biotechnology related subject and this PPT contais all blotting techniques in a very elaborative concise manner includes procedure principle application etc so which itwould help any bio student to take proper knowledge in this topic. I hope you will enjoy the content of the topic and would be able to grasp the topic properly

1. Blotting Techniques B.Sc (Life Science) 3 rd Year : Rohit Mondal B.Sc (Life Science) 3 rd yr Sri Aurobindo College DSE-Plant Analytical technique

2. What is a blot ? In molecular biology blots is a technique for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of gel electrophoresis. Types Of Bloting : Southern Blotting : Used For DNA Detection in a sample Northern Blotting : Used For RNA Detection in a sample Western Blotting : Used For Proteins in a sample

3. Southern Blotting A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British biologist Edwin Southern. Other blotting methods (i.e.,  western blot ,  northern blot ,  eastern blot ,  southwestern blot ) that employ similar principles, but using RNA or protein, have later been named in reference to Edwin Southern’s name.

4. Principle of Southern Blotting Southern blotting is a hybridization technique for identification of particular size of  DNA  from the mixture of other similar molecules .  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.

5. Procedure

6. Procedure Restriction digest: by RE enzyme and amplification by PCR Gel electrophoresis: SDS gel electrophoresis Denaturation : Treating with HCl and NaOH Blotting Baking and Blocking with casein in BSA Hybridization using labelled probes Visualization by autoradiogram

7. Southern Blotting

8. Southern Blotting

9. Application of Southern Blotting Southern blotting technique is used to detect DNA in given sample. DNA finger printing is an example of southern blotting Used for paternity testing, criminal identification, victim identification To isolate and identify desire gene of interest. Used in restriction fragment length polymorphism To identify mutation or gene rearrangement in the sequence of DNA Used in diagnosis of disease caused by genetic defects Used to identify infectious agents

10. Southern Blotting https://www.youtube.com/watch?v=TOKhHy7rU18

11. What is a Northern Blotting ? The northern blotting is a technique used in molecular biology research to study gene expression by detection of RNA. The Northern blot, also known as the RNA blot, is one of the blotting techniques used to transfer RNA onto a carrier for sorting and identification. The Northern blot is similar to the Southern blot except that RNA instead of DNA is the subject of analysis in this technique. It is mRNA which is isolated and hybridized in northern blots.

12. Discovery of Northern Blotting The northern blot technique was developed in 1977 by James Alwine , David Kemp, and George Stark at Stanford University.  J.C. Alwine , a biologist with a sense of humor, developed a technique analogous to the Southern blot, this time for the identification of a specific RNA within a complex RNA sample using a radio- labelled DNA probe. Alwine couldn’t resist the temptation to call his technique the  northern blot  in an allusion to Southern’s technique, raising a chuckles in labs everywhere.

13. Principle of Northern Blotting Northern blotting is a method used to study gene expression by detection of RNA in a sample. Therefore,it is also called RNA Blot . The sample RNA is isolated from an organism of interest and then electrophoresed on agarose gel which separates the fragments on the basis of their size. The separated RNA fragments are transferred to a support membrane(nitrocellulose membrane). This can be performed by simple capillary method in presence of a specific buffer.

14. The RNA is then immobilized on membrane eitherby baking at high temperature or UV crooslinking,which results in covalent linkage of RNA to membrane preventing nucleic acid from being washed away from subsequent processing.

15. This is followed by hybridisation with a labeled DNA or RNA probe. If the sample contains the complementary RNA sequence, the probe will bind to membrane to form double stranded DNA-RNA hybrid molecule between single stranded DNA probe and single stranded target RNA . The final step is the detection of RNA of interest on the membrane using chromogen .

16. Requirements of Northern Blotting Agarose Gel for process of gel electrophoresis . Nylon membrane/ Diazo benzyl oxy methyl (DBM) filter paper . Complementary Radioactive probe for hybridisation . Formaldehyde(HCHO) for degradation - Carbonal group reacts to form stiff base with amino group of GAC, this prevents normal H-bonding & Hence maintain RNA in denatured State. X-ray film for identification of RNA. Note -No need of restriction enzyme for Northern Blotting.

17. Procedure Step 1 : DNA containing the gene of interest is exteacted from human cells and cut into fragments by ristriction enzymes. Step 2 : The fragments are separated According to size by gel electrophoresis. Each band consist of many copies of a particular DNA fragment. Thus bands are invisible but can be visible by straining. Step 3 : The DNA bands are transferred to a nitrocellulose filter by blotting. The solution passes through the gel and filter to the paper towels.

18. Schematic view of Northern Blot kk

19. Step 4 : This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel. Step 5 : The filter is exposed to radioactivity labelled probe for a specific gene. The Probe will base- pair ( hybridise ) wiyh a short sequence present on the gene. Step 6 : The filter is then exposed to X ray Film. The Fragment containing the gene of interest is identified by a band on the developed film.

20. Application of Northern Blotting Observe a particular gene's expression,pattern between tissues,organs , development stages,environment stress levelS etc. Used to show overexpression of oncogenes and down regulation of tumour suppressor gene's in cancerous cells. Detecting a specific mRNA in sample used for screening recombinant which are successfully transformed with transfer. Also used for studying mRNA Splicing.

21. Reverse Northern Blotting The reverse northern blot is a method by which gene expression patterns may be analyzed by comparing isolated RNA molecules from a tester sample to samples in a control cDNA library. It is a variant of the northern blot in which the nucleic acid immobilized on a membrane is a collection of isolated DNA fragments rather than RNA, and the probe is RNA extracted from a tissue and radioactively labelled .

22. Advantages of Northern Blotting Northern blots are particularly useful to determine conditions under which specific genes are expressed . Only mRNA from cell types that are synthesizing protein will hybridize to the probe . It is also useful in detection of mRNA transcript size.Specifity is relatively high. Blots can be stored for several years and reprobed if necessary.

23. Disadvantages of Northern Blotting Risk of mRNA degradation during electrophoresis: quality and quantification of expression are negatively affected . High doses of radioactivity and formaldehyde are a risk for workers and the environment Detection with multiple probes is difficult and also time consuming procedure Use of ethidium bromide, DEPC and UV light needs special training and attention.

24. Precaution used in Northern blotting Remove air bubbles trapped between the gel & the membrane. Ensure that all buffer components are fully dissolved before using. Ensure that the electrophoresis tanks are rinsed with distilled water after used. Control the temperature during hybridization. Always check for the incorporation of radioactive label before using the probe. Don’t reuse electrophoresis buffer & radioactive probes.

25. Western Blotting: Definition & Principle Western blotting is a technique which is used for identification of particular protein from the mixture of protein. In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test. Western blotting is also known as immunoblotting because it uses antibodies to detect the protein

26. Procedure Extraction of protein Gel electrophoresis: SDS PAGE Blotting: electrical or capillary blotting Blocking: BSA Treatment with primary antibody Treatment with secondary antibody( enzyme labelled anti Ab ) Treatment with specific substrate; if enzyme is alkaline phosphatase , substrate is p-nitro phenyl phosphate which give color.

27. Application To determine the size and amount of protein in given sample. Disease diagnosis: detects antibody against virus or bacteria in serum. Western blotting technique is the confirmatory test for HIV. It detects anti HIV antibody in patient’s serum. Useful to detect defective proteins. For eg Prions disease. Definitive test for Creutzfeldt -Jacob disease, Lyme disease, Hepatitis B and Herpes

28. Western Blotting https:// www.youtube.com/watch?v=GJJGNOdhP8w https:// www.youtube.com/watch?v=JcN0EkcHrKk https://www.youtube.com/watch?v=IoVzpL_heFo

29. Precaution Avoid buying cheap antibodies Be careful when re-using antibodies Dissolve the milk (or blocking buffer) properly to avoid black dots Use an internal standard to ease quantification between different membranes

30. THANK YOU