Question

‘There is a paternity dispute for a child’. Which technique can solve the problem. Discuss the principle involved.

Answer 1

DNA finger printing is used to solve the paternity dispute. DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called as repetitive DNA, because in these sequences, a small stretch of DNA is repeated many times. These repetitive DNA are separated from bulk genomic DNA as different peaks during density gradient centrifugation.

• The bulk DNA forms a major peak and the other small peaks are referred to as satellite DNA. Depending on base composition (A : T rich or G : C rich), length of segment, and number of repetitive units, the satellite DNA.
• DNA is classified into many categories, such as micro-satellites, mini-satellites etc. These sequences normally do not code for any proteins, but they form a large portion of human genome.
• These sequence show high degree of polymorphism and form the basis of DNA fingerprinting. Since DNA from every tissue (such as blood, hair- follicle, skin, bone, saliva, sperm etc.), from an individual, show the same degree of polymorphism, they become very useful identification tool in forensic applications. Further, as the polymorphisms are inheritable from parents to children, DNA fingerprinting is the basis of paternity testing, in case of disputes. 
• The technique of DNA fingerprinting was initially developed by Alec Jeffreys. Lalji Singh is called father of Indian DNA fingerprinting or DNA profiling or DNA typing. He used a satellite DNA as probe that shows very high degree of polymorphism. It was called as Variable Number of Tandem Repeats (VNTR).
• The technique, as used earlier, involved Southern blot hybridisation using radiolabelled VNTR as a probe. It included 

1. Isolation of DNA,
2. Digestion of DNA by restriction endonucleases,
3. Separation of DNA fragments by electrophoresis,
4. Transferring (blotting) of separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon,
5. Hybridisation using labelled VNTR probe,
6. Detection of hybridised DNA fragments by autoradiography.