1.
Obtaining DNA (Isolation/Extraction) : The first step is to obtain a
sample of DNA from blood, saliva, hair roots, semen etc. If needed many copies
of the DNA can be produced by PCR (cloning/DNA amplification)
2.
Fragmenting DNA (Restriction Digestion) : Treating DNA with Restriction Enzymes
(Restriction endonucleases) which cut the DNA into smaller fragments by
cutting it at specific sites.
3.
Separation of DNA fragments by electrophoresis : DNA fragments are applied at one
end of agarose
gel plate.
When an electric current is applied to the gel, the DNA fragments (which are
slightly negatively charged) travel across the gel (smaller and more mobile
pieces travel farther). This technique of separation of DNA fragments into individual
bands is called Gel Electrophoresis.
4.
Denaturing DNA : The DNA on the gel is 'denatured' using alkaline
chemicals or by heating, (denaturing means separation/splitting of the double
helix into 'single strands' by breaking hydrogen bonds between the two
strands).
5.
Blotting : A thin nylon membrane is placed over the 'size fractionated DNA strands' and covered by paper
towels. As the towels draw moisture the DNA strands are transferred on to the
nylon membrane by capillary action. This process is called 'Blotting' - more
precisely Southern
blotting, after
the name of its inventor E.M. Southern.
6.
Using probes to identify specific DNA: A radioactive
probe (DNA is
labeled with a radioactive substance) is added to the DNA bands. The Probe is a single
stranded DNA
molecule
that is 'complementary' to the gene of interest in the sample under study. The
probe attaches by base pairing to those restriction fragments that are
complementary to its sequence. The probes can be prepared by using either
'fluorescent substances' or 'radioactive isotopes'.
7.
Hybridisation with probe: After the probe hybridises and
the excess probe washed off, a photographic film is placed on the membrane
containing 'DNA hybrids'.
8.
Exposure on film to make a Genetic/DNA Finger Print: The radioactive label exposes
the film to form an image (image of bands) corresponding to specific DNA bands.
The thick and thin dark bands form a pattern of bars which constitute a Genetic
fingerprint
