In Southern Blotting, Specific DNA Sequences Are Detected And Isolated From Complex Mixtures Of DNA Fragments

Southern Blotting

Southern Blotting is a laboratory technique used to detect and isolate a specific DNA sequence from a complex mixture of DNA fragments. It was first introduced in 1975 by Dr. Edwin Southern, who later received a Nobel Prize in Physiology or Medicine in 1980 for his contribution to the field of molecular biology.

The Southern Blot technique involves four major steps: DNA digestion, gel electrophoresis, DNA transfer, and hybridization.

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DNA Digestion:

In the first step, the DNA sample is digested with a restriction enzyme, which is a type of enzyme that recognizes a specific sequence of DNA and cleaves the DNA at that site. The restriction enzyme cuts the DNA into smaller fragments, and each fragment contains a different sequence of DNA.

Gel Electrophoresis:

In the second step, the DNA fragments are separated according to their size using gel electrophoresis. The DNA fragments are loaded onto a gel, which is a thin, flat sheet made of agarose, a complex carbohydrate. An electric field is applied across the gel, and the negatively charged DNA fragments move towards the positive electrode. The smaller fragments move faster and travel further down the gel than the larger fragments.

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DNA Transfer:

In the third step, the separated DNA fragments are transferred from the gel to a nylon or nitrocellulose membrane. This process is called Southern Blotting. The membrane is placed on top of the gel and covered with absorbent paper or sponge. A weight is placed on top of the stack to ensure good contact between the gel and the membrane. The DNA fragments move out of the gel and onto the membrane by capillary action.

Hybridization:

In the final step, the membrane is hybridized with a labeled DNA probe that is complementary to the specific DNA sequence of interest. The probe is a short single-stranded DNA fragment that has been labeled with a radioactive or fluorescent molecule. The probe binds to the complementary DNA sequence on the membrane, forming a hybrid or double-stranded DNA molecule. The location of the hybrid molecule on the membrane can be visualized by autoradiography or fluorescence imaging.