Southern Blotting

The Southern Blot is the method to analyze the genetic patterns in the DNA. The protocol of Southern Blot involves:

Preparation of DNA sample

Isolation of DNA

Isolating the DNA of interest from the Nucleus. This can be done either chemically, by using a detergent to wash the extra material from the DNA, or mechanically, by applying a large amount of pressure in order to “squeeze out” the DNA.

DNA cleaving
Cutting the DNA into several pieces of different sizes. This is done using one or more restriction enzymes.

Size Fractionation
Sorting the DNA pieces by size. The process by which the size separation, “size fractionation,” is done is called gel electrophoresis.

Gel Electrophoresis
The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel. The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.

Denaturing the DNA, so that the entire DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel.

Blotting the DNA
The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper, and then baked to permanently attach the DNA to the sheet for southern blotting.

Southern blotting

In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA of interest. If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bond with the denatured DNA on the paper, only the areas where the radioactive probe binds will show up on the film. This allows researchers to identify, in a particular person’s DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.

Preparation of Probe
Preparation of Radioactive Probe involves the following procedure

1. Mix the DNA with Endonuclease enzyme in a test tube.
2. Endonuclease will break the DNA into segments.
3. Add nucleotides to the nicked DNA one nucleotide should be radio labeled.
4. Add DNA polymerase so that it will heal the Brocken segments of DNA. During this repair, radioactive nucleotide will be incorporated in the DNA.
5. The nicked DNA is then heated, splitting the two strands of DNA apart. This creates single-stranded radioactive and non-radioactive pieces. The radioactive DNA, now called a probe is ready for use.

DNA Hybridization

Hybridization is the binding, of two genetic sequences.

1. Before hybridization, DNA must first be denatured, using heat or chemicals.
2. Denaturing results in the production of single stranded DNA.
3. Once the DNA has been denatured, a single-stranded radioactive probe can be used to check whether the denatured DNA contains a nucleotide sequence similar to that on the probe.
4. The denatured DNA is then thoroughly shaken with some saline to bind the probe firmly with the DNA.

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Southern Blotting

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