Blotting techniques and types of blotting .pptx

Blotting techniques- types and advantages, disadvantages

1. A presentation on BLOTTING TECHNIQUES Presented by: Under guidance of: Ms. Bhongal Sakshi Adinath Prof. V.V. Nimbalkar F.Y.M.Pharm M.Pharm Roll no- 03 Dept. of Pharmacology Dept. of Pharmacology Dr.V.V.P.F’s College Of Pharmacy, Dr.V.V.P.F’s College of Pharmacy, Vilad ghat , Ahmednagar Vilad Ghat , Ahmednagar 1

2. CONTENTS : Introduction Southern blotting Northern blotting Dot-blotting Western blotting Colony and plaque blotting 2

3. Blotting techniques are very widely used analytical tools for the specific identification of desired DNA or RNA fragments from thousands of molecules. Blotting refers to the process of immobilization of sample nucleic acids or solid support (nitrocellulose or nylon membranes). The blotted nucleic acids are then used as targets in the hybridization experiments for their specific detection INTRODUCTION : 3

4. Fig no 1: Diagrammatic representation of typical blotting apparatus 4

5. The most commonly used blotting techniques are listed below - Southern blotting (for DNA) Northern blotting (for RNA) Western blotting (Protein) Dot blotting (DNA/RNA) Colony and plaque hybridization (cells from colony). 5

6. SOUTHERN BLOTTING : Southern blotting technique is the first nucleic acid blotting procedure developed in 1975 by Southern Genomic DNA isolated from cells digested with one or more restriction enzymes This mixture is loaded into a well in an agarose polyacrylamide gel and then subjected to electrophoresis. DNA, being negatively charged migrates towards the anode (positively charged electrode); the smaller DNA fragments move faster. The separated DNA molecules are denatured by exposure to a mild alkali and transferred to nitrocellulose or nylon paper. Fig no. 2 :Diagrammatic representation of southern blotting 6

7. This results in an exact replica of the pattern of DNA fragments on the gel The DNA can be annealed to the paper on exposure to heat (80°C The nitrocellulose or nylon paper is then exposed to labelled cDNA probes. These probes hybridize with complementary DNA molecules on the paper Paper after thorough washing is exposed to X-ray film to develop autoradiograph. This reveals specific bands corresponding to the DNA fragments recognized by cDNA probe. 7

8. Applications of Southern blotting- It is an invaluable method in gene analysis Important for the confirmation of DNA cloning results Useful for mapping restriction sites around single copy gene sequence Forensically applied to detect minute quantities of DNA (to identify parenthood, thieves, rapists Highly useful for the determination of restriction fragment length polymorphism (RFLP) associated with pathological conditions. DNA pieces from one species (e.g. Human) can be used to detect DNA molecules from related species (e.g. Chimpanzee, cow). This technique is referred to as Zoo-blotting 8

9. NORTHERN BLOTTING : Northern blotting is the technique for the specific identification of RNA molecules RNA molecules are subjected to electrophoresis, followed by blot transfer, hybridization and autoradiography RNA molecules do not easily bind to nitrocellulose paper or nylon membranes Blot- transfer of RNA molecules is carried out by using a chemically reactive paper prepared by diazotization of aminobenzyloxymethyl to create sites around a diazobenzyloxymethyl (DBM) paper The RNA can covalently bind to DBM paper. Fig no.3 : Diagrammatic representation of northern blotting 9

10. The blot-transferred RNA molecules hybridize with DNA probes which can be detected autoradiography Northern blotting is theoretically, a good technique for determining the number of genes (through mRNA) present on a given DNA. But this is not really practicable since each gene may give rise to two or more RNA transcripts Another drawback is the presence of axons and introns. 10

11. APPLICATION OF NOTHERN BLOTTING- A standard for the study of gene expression at the level of mRNA Study of RNA degradation. Detection of mRNA transcript size. Study of RNA half life. In gene expression studies. Blots can be stored for several years and reprobed if necessary 11

12. DOT-BLOTTING : Dot blotting is a modification of southern and northern blotting techniques In this approach , nucleic acids ( DNA or RNA ) are directly spotted onto the filters , and not subjected to electrophoresis . The hybridization procedure is same as in original blotting techniques. Dot-blotting technique is particularly useful in obtaining quantitative data for evaluation of gene expression. 12

13. WESTERN BLOTTING : Western blotting involves the identification of proteins. It is to very useful to understand the nucleic acid functions, particularly during the course of gene manipulations. The technique of Western blotting involves the transfer of electrophoreses protein bands from polyacrylamide gel to nylon or nitrocellulose membrane These proteins can be detected by specific protein-ligand interactions. Antibodies or lectins are commonly used for this purpose 13

14. Application of western blotting- To determine the size and amount of protein in sample Western blotting technique is confirmatory test for HIV. It detects anti HIV antibody inpatients serum Useful to detect defective proteins. For ex.- Prion disease. Disease diagnosis: detects antibodies against virus or bacteria in serum. Definitive test for hepatitis B , herpes. 14

15. COLONY AND PLAQUE BLOTTING : Colony and plaque blotting is a process of hybridization for specific identification and purification of colonies. The desired bacteria are grown as colonies on an agar plate . When a nitrocellulose filter paper is overlaid on the agar plate , colonies get transferred. They permanently fixed to the paper by heat. On treatment with alkali , the cells lyse and DNA gets denatured . When these DNA prints are exposed to specific probes hybridization occurs. Fig no. 4: Diagrammatic representation of colony and plaque blotting 15

16. REFERENCES : U. Satyanarayana , Biotechnology ,8 th edition , Books and allied (P) Ltd. 2012 , p.:97-101 Hayes , Peter,. Et al. “Blotting Techniques For The Study Of DNA, RNA ,AND Proteins’’. BMJ: British Medical Journal , vol.299, no. 6705, 1989, p.965-968. JSTOR Loudiya, Krishna and Mani , Arghya. (2021) . Southern Blotting Technique. 3. p- 134-136 Mahmood T, Yang PC. Western blot :technique, theory, and trouble shooting . N m J Med Sci.2012 Sep;4(9): 429-34. Kurien BT , Scofield RH , Oklahoma City OK. Electrophoresis – blotting techniques. Elsevier reference module in chemistry , molecular sciences and chemical engineering . Elsevier , Waltham, MA. Doi.2015; 10 16

17. THANK YOU. 17