Question

Explain the isolation of gene of interest(by electrophoresis).

Answer 1

Electrophoresis is a method of separating DNA fragment according to their size and molecular weight.

1. The cell containing gene of interest is taken.

2. The genome is isolated from the cell by lysis method.

3. The fragment of genome containing gene of interest is obtained using restriction endonuclease enzyme. The restriction enzyme cuts the DNA at a particular site.

4. Electrophoresis: The DNA fragment is then separated based on the size and molecular weight on electrophoresis. The samples are loaded in the well of the gel. One well is kept for a DNA ladder that contains DNA fragments of known lengths. After this, the gel is subjected to the electric current. The DNA molecules are negatively charged because of the phosphate groups in the sugar-phosphate backbone. Hence, they will move through the matrix of the gel towards the positive pole. The large fragment of DNA will be near the negative pole and smallest fragments of DNA will be near the positive pole.

5. Once, the gene of interest is separated on electrophoresis, southern blotting is carried out. In this, the DNA is transferred to the nitrocellulose paper. It is then hybridized with the radioactive probe and seen using X-ray.