Question

Enlist the steps in DNA fingerprinting giving its applications.

Answer 1

Steps in DNA fingerprinting:

1. Isolation of DNA:
   DNA can be isolated from small amounts of tissue such as blood, hair roots, skin, and so on.
2. Restriction digestion:
   
   1. The recovered DNA is next processed with restriction enzymes, which cut the DNA at specified locations, resulting in little fragments of varying lengths.
   2. Restriction fragment length polymorphism (RFLP) refers to variations in the length of restriction fragments.
3. Gel electrophoresis: 
   
   1. The DNA samples are placed onto an agarose gel and electrophoresed.
   2. DNA fragments with negative charges migrate to the positive pole.
   3. Fragment separation is determined by fragment length and results in the creation of bands.
   4. Alkali treatment is then used to denature ds DNA in ssDNA.
4. Southern blotting: 
   The DNA fragments are isolated and put into a nylon or nitrocellulose membrane.
5. Selection of DNA probe:
   
   1. A DNA probe is a known single-stranded DNA sequence.
   2. It is derived from organisms or created using the cDNA production procedure.
   3. Radioactive isotopes are used to label the DNA probe.
      
6. Hybridization: 
   
   1. A probe is introduced to a nitrocellulose membrane containing DNA fragments during this process.
   2. The single-stranded DNA probe binds to the DNA strand's corresponding base sequence.
   3. As a result, on the nitrocellulose membrane, DNA-DNA hybrids occur. Single-stranded DNA probe fragments that have not been bound are washed away.
      
7. Photography: 
   The nitrocellulose membrane is then maintained in contact with the X-Ray film. Due to the radioactive probe, DNA bands produce a photographic image on X-ray film. This is called autoradiography.