Question

Explain different steps involved in DNA Fingerprinting.

Answer 1

The steps of the DNA fingerprinting technique are as follows:

1. Isolation of DNA: The DNA must be isolated from the cells or tissues of the body (host). An only a small amount of tissue-like blood, hair roots, skin, etc. is required.
2. Restriction digestion: The isolated DNA is treated with restriction enzymes. The restriction enzymes cut the DNA into small fragments having variable lengths. This phenomenon is called Restriction Fragment Length Polymorphism (RFLP).
3. Gel electrophoresis: The DNA samples are loaded for agarose gel electrophoresis under an electric influence. The DNA fragments, which are negatively charged move to the positive pole. The movement of these fragments depends on the length of fragments. This results in the formation of bands. dsDNA splits into ssDNA by alkali treatment.
4. Southern blotting: The separated DNA fragments are transferred to a nylon membrane or a nitrocellulose filter paper by placing it over the gel and soaking them with filter paper overnight.
5. Selection of DNA probe: A known sequence of single-stranded DNA is prepared. It is called DNA Probe. DNA Probe is obtained from organisms or prepared by the cDNA preparation method. The DNA probe is labelled with radioactive isotopes.
6. Hybridization: Probe DNA is added to the nitrocellulose filter paper containing host DNA. The single-stranded DNA probe pairs with the complementary base sequence of the host DNA strand. As a result, DNA-DNA hybrids are formed on the nitrocellulose filter paper. The remaining single-stranded DNA probe fragments are washed off.
7. Photography: The nitrocellulose filter paper is photographed on an X-ray film by autoradiography. The film is analyzed to determine the presence of hybrid DNA.