Southern blotting technique

Blots are techniques for transferring DNA, RNA and proteins onto a solid support (carrier) generally nylon or nitrocellulose membranes. so they can be separated, and often follows the use of a gel electrophoresis

1. 8/30/2018 By Hirut G. 1 Southern Blotting technique By : Hirut G/Michael( DVM,Lecturer )   April, 2018 Bishoftu , Ethiopia            

2. S OUTHERN BLOTTING Technique 8/30/2018 By Hirut G. 2

3. Outlines of this presentation  Blotting  Types of blotting  Southern blotting  Principle  Apparatus 8/30/2018 By Hirut G. 3

4.  Steps involved in Southern blotting  A schematic view of Southern blotting  Application  Advantages and Disadvantages of Southern blotting 8/30/2018 By Hirut G. 4

5. What is blotting? Blots are techniques for transferring DNA, RNA and proteins onto a solid support (carrier) generally nylon or nitrocellulose membranes. so they can be separated , and often follows the use of a gel electrophoresis . 8/30/2018 By Hirut G. 5

6. Blotting of nucleic acid is the central technique for hybridization studies. Nucleic acid labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving understanding of gene expression, organization, etc. 8/30/2018 By Hirut G. 6

7. Identifying and measuring specific proteins in complex biological mixtures, such as blood, have long been important goals in scientific and diagnostic practice . 8/30/2018 By Hirut G. 7

8. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and genetic counseling. Blotting techniques are used to identify unique proteins and nucleic acid sequences. 8/30/2018 By Hirut G. 8

9. They have been developed to be highly specific and sensitive and have become important tools in both molecular biology and clinical research. 8/30/2018 By Hirut G. 9

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12. 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. 8/30/2018 By Hirut G. 12

13. The method is named after its inventor, the British biologist Edwin Mellor Southern. 8/30/2018 By Hirut G. 13

14. The key to this method is hybridization . Hybridization is a process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA. 8/30/2018 By Hirut G. 14

15. There are 2 important features of hybridization: The reactions are specific-the probes will only bind to targets with a complementary sequence. The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules . 8/30/2018 By Hirut G. 15

16. 1. Extract and purify DNA from cells; 2. DNA is restricted with enzymes;; 3 . Denature DNA; 4 Separated by electrophoresis ; 5. Transfer to nitrocellulose paper; 6. Add labeled probe for hybridization to take place; 7. Wash off unbound probe; 8. Autoradiograph. Steps for hybridization 8/30/2018 By Hirut G. 16

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18. 3.The restriction fragments present in the gel are denatured with alkali and transferred onto 4. a nitrocellulose filter or nylon membrane by blotting. This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter. 8/30/2018 By Hirut G. 18

19. 5. The filter is incubated under hybridization conditions with a specific radiolabeled DNA probe. The probe hybridizes to the complementary DNA restriction fragment. 8/30/2018 By Hirut G. 19

20. This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter. 8/30/2018 By Hirut G. 20

21. Excess probe is washed away and the probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized. 8/30/2018 By Hirut G. 21

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26. Southern blots are used in gene discovery , mapping, To identify specific DNA in a DNA sample. To Isolate desired DNA for construction of rDNA . Identify mutations, deletions, and gene rearrangements . Used in prognosis of cancer and in prenatal diagnosis of genetic diseases. 8/30/2018 By Hirut G. 26

27. In RFLP ( in molecular biology; Southern blot analysis is used for the detection of DNA or gene sequence in circular or large DNA. ... Where as RFLP (Restriction Fragment Length Ploymorphism ) is used for compare the DNA fragments after reaction with restriction enzymes. 8/30/2018 By Hirut G. 27

28. Diagnosis of HIV-1 and infectious disease. In DNA fingerprinting: Paternity and Maternity Testing Criminal Identification and Forensics Personal Identification 8/30/2018 By Hirut G. 28

29. Effective way to detect a specific DNA sequence in a large , complex sample of DNA. Can be used to quantify the amount of the present DNA. Cheaper than DNA sequencing 8/30/2018 By Hirut G. 29

30. Southern blots allow investigators to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples. 8/30/2018 By Hirut G. 30

31. More expansive than most other tests. Complex and labor-intensive. Time consuming and cumbersome . 8/30/2018 By Hirut G. 31

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