Question

Explain different steps involved in DNA Fingerprinting.

Write the steps carried out in the process of DNA fingerprinting technique.

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.

Answer 2

Steps involved in DNA fingerprinting:

• DNA is first extracted from cells such as those found in blood stains, semen stains, or hair roots.
• The isolated DNA is then amplified (copied many times) using the polymerase chain reaction (PCR).
• The DNA is cut into fragments using restriction enzymes (restriction endonucleases).
• These DNA fragments are separated according to size by gel electrophoresis.
• The separated fragments are transferred (blotted) onto a synthetic membrane like nylon or nitrocellulose and baked at about 80°C for 3–5 hours to fix the DNA onto the membrane.
• The double-stranded DNA is converted into single-stranded DNA.
• Hybridisation is carried out using labelled VNTR probes.
• The hybridised DNA fragments are detected through autoradiography. This produces bands of different sizes, forming a unique pattern that varies from person to person.
• The pattern of dark and light bands obtained is known as the DNA fingerprint.
• Even DNA from a single cell is sufficient to carry out DNA fingerprinting.