southern blotting.pptx

Blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.
Types of blotting techniques
Southern Blotting
Northern Blotting
Western 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 Mellor Southern.
- Methods in Southern blotting
- Advantages and disadvantages

1. SOUTHERN BLOTTING PRESENTED BY: NUSRAT SHEIKH

2. Content Introduction to Blotting Types of blotting Southern blotting Principle Methods involved in southern blotting A schematic view of southern blotting Application Advantages and Disadvantages Reference

3. Blotting A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier. The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.

4. Types of blotting techniques 1 ) Southern blotting ( to detect DNA ) 2 ) Northern blotting ( to detect RNA ) 3 ) Western blotting ( to detect protein )

5. 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 Mellor Southern .

6. Principle This method Involves separation, transfer and hybridization. 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. 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.

7. Methods in Southern blotting Restriction endonucleases cut high-molecular-weight DNA strands into smaller fragments. The mixture of fragmented DNAs fig: Restriction endonuclease is separated in agarose get electrophoresis. The restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting. fig: Gel Electrophoresis

8. This procedure preserves the distribution of the fragments in the gel creating a replica of the gel on the filter. A sheet of nitrocellulose or nylon membrane is placed on top of the Fig: DNA denaturation by NaOH gel. Pressure is applied evenly to the gel to ensure good and even contact between gel and membrane. Fig: Membrane is placed on top of gel Cont..

9. Cont.. Membrane is then baked in a vacuum or regular oven at 80°C for 2 hours or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane. The probe is added to the matrix to bind to the molecules. Any unbound probes is washed away and the probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized.

10. Prehybridization This step involved in closing the binding site of membrane other than DNA fragments. For this a special prehybridization solution is used. Such as- 1.polyvinylpyrrolidone 2.bovine serum albumin 3.dried milk After this, membrane is washed and ready for hybridization

11. A Schematic view of southern blotting

12. Applications 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. In RFLP, gene mapping and discovery. Diagnosis of HIV-1 and infectious disease. In DNA fingerprinting: -Paternity and Maternity Testing -Criminal Identification and Forensics -Personal Identification

13. Advantages 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 Disadvantages More expensive than other tests Complex and labour intensive Time consuming and cumbersome Require large amount of DNA

14. Reference S.P. Vyas., Pharmaceutical Biochemistry, CBS Publishers, New Delhi,2008. U. Satyanarayana, U. Chakrapani, Biochemistry, Arunabha Sen Books Pvt.Ltd., 2010. Robert K. Murray, Daryl K. Granner, Peter A. Mayer, Victor W. Rodwell., Harper’s Illurstrated Biochemisry, Lange Medical publications, 2005 http://sciencing.com

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