Arrange the following steps in order for the technique of DNA finger printing.

A. Isolation of DNA.

B. Separation of DNA fragments by gel, electrophoresis.

C. Digestion of DNA by restriction endonucleases.

D. Blotting of separated DNA fragments to nylon membrane.

E. Hybridisation using VNTR probe followed by autoradiography.

Choose the correct answer from the options given below:

A,C,B,D,E

The correct answer is Option (1) –A,C,B,D,E

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.

The technique of DNA Fingerprinting was initially developed by Alec Jeffreys. 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) detection of hybridised DNA fragments by autoradiography.