Question

Mention the significance of gel electrophoresis in r- DNA technology?

Answer 1

Hint: Recombinant DNA is the DNA formed by combining DNAs from different organisms.
Recombinant DNA technology involves two basic processes -
(i) formation of recombinant DNA and
(ii) introduction of recombinant DNA into an appropriate host.

Complete answer: DNA has to be isolated in pure form for the action of restriction enzymes.
1. Fragmentation of DNA is carried out by incubating the purified DNA molecules with suitable restriction enzymes at optimal conditions of temperature and pH
2. The fragments of DNA are separated by a technique called gel electrophoresis.
The DNA is cut into fragments by a restriction endonuclease.
These fragments are separated by gel electrophoresis.
3. Agarose, a natural polymer obtained from seaweed is used as the matrix.
4. DNA fragments being negatively charged are separated by forcing them to move through the matrix towards the anode under an electric field.
5. The DNA fragments separate/resolve according to their size.
6. The separated molecules are stained by ethidium bromide and visualized by exposure to UV radiation, as bright orange colored bands.
7. The separated DNA ( on the gel ) is cut from the gel and extracted from the gel piece (elution). Such DNA fragments are purified and used for constructing recombinant DNA by joining them with cloning vectors.

Additional information: Process of recombinant DNA technology involves the following steps:
1. Isolation of DNA
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of the desired DNA fragment
4. Amplification of the gene of interest
5. Ligation of the DNA fragment into a vector using DNA ligase
6. Transfer of recombinant DNA into the host
7. Culturing the host cells on a suitable medium on a large scale
8. Extraction of the desired product.
9. Downstream processing of the product as a finished product ready for marketing.

Note: The properties of DNA that help in the formation of recombinant DNA are denaturation and renaturation.
Denaturation is the separation of two strands by breaking hydrogen bonds on heating and renaturation is the reunion of the complementary strand to form DNA duplex cooling.