Anushi JainRoll No : 12

Paper 3MSc-I

SOUTHERN HYBRIDIZATIO

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Contents Blotting Types of blotting Southern blotting Principle Apparatus Steps involved in southern blotting A schematic view of southern blotting Application Advantages and Disadvantages

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.

• Technique for transferring DNA ,RNA and Proteins onto a carrier so they can be separated, and often

follows the use of a gel electrophoresis.

Types Of BlottingSouthern blotting

Northernblotting

Westernblotting

Southern Blotting• A Southern blot is a method used

in molecular biology for DNA analysis.

•The method is named after its inventor, the British biologist Edwin Mellor Southern,In 1975.

•This method is also known as DNA blotting/Southern hybridization.

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.

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.

APPARATUS

Steps Involved In Southern Blotting

1. Extract and purify DNA from cells;

2. DNA is restricted with enzymes;

3. Separated by electrophoresis;

4. Denature DNA;

5. Transfer to nitrocellulose paper;

6. Add labelled probe for hybridization to take place;

7. Wash off unbound probe;

8. Autoradiograph.

1. Extract and purify DNA from cells;

2. DNA is restricted with enzymes;

3. Separated by electrophoresis;

4. Denature DNA;5. Transfer to nitrocellulose paper;

6. Add labeled probe for hybridization to take place;7. Wash off unbound probe;8. Autoradiograph.

DNA Fragments seen after autoradiography

A Schematic View Of Southern Blotting

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 prenatal diagnosis of genetic diseases.• In RFLP.• In DNA fingerprinting:

Paternity and Maternity Testing Criminal Identification and Forensics

 

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• Complex and labor-intensive.

• Time consuming.