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

$DNA$ 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 $DNA$

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

$(i)$ Isolation of $DNA$, $(ii)$ Digestion of $DNA$ by restriction endonucleases, $(iii)$ Separation of $DNA$ fragments by electrophoresis, $(iv)$ Transferring (blotting) of separated $DNA$ fragments to synthetic membranes, such as nitrocellulose or nylon,

$(v)$ Hybridisation using labelled $VNTR$ probe, and $(vi)$ Detectionof hybridised $DNA$ fragments by autoradiography.