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. These sequences show high degree of polymorphism (variation at genetic level) and form the basis of DNA Fingerprinting.
Polymorphism can be defined as, an inheritable mutation is observed in a population at high frequency, it is referred to as DNA polymorphism.
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 Variable Number of Tandem Repeats (VNTRs).
Mechanism of DNA Fingerprinting
Extraction : DNA is extracted from the small amounts of blood, semen or hair bulbs available.
Amplification : Many copies of this DNA are made by a technique called Polymerase Chain Reaction (PCR).
Restriction Digestion : DNA is cut into desired reproducible segments using restriction enzymes.
Separation : These DNA sequences (restriction fragments) are separated by gel electrophoresis.
Southern Blotting : The separated DNA sequences are transferred from gel onto a nitrocellulose membrane.
Hybridisation with probe, the DNA sequence complementary to VNTR sequences.
Exposure of the membrane to X-ray film, whose specific bands are developed.
It is used effectively in forensic science for identifying :
(a) the biological father (in case of paternity disparity)
(b) the criminals such as murderers and rapists.
HUMAN GENOME PROJECT
The Human Genome Project was a 13-year project coordinated by the U.S. Department of Energy and the National Institute of Health, additional contributions came from Japan, France, Germany, China and other. The project was completed in 2003.
The methods involved two major approaches. One approach focused on identifying all the genes that expressed as RNA referred as Expressed Sequence Tags (ESTs). The other approach is blind approach of simply sequencing the whole set of genome that contained all the coding and non-coding sequences and later assigning different regions in the sequence with functions, referred as sequence Annotation.
Steps Involved in Sequencing
(a) Isolation of total DNA from a cell and converted into random fragments.
(b) Cloning of DNA fragments can be performed by using cloning vectors like BAC (Bacterial Artificial Chromosomes) and YAC (Yeast Artificial Chromosomes).
(c) The fragments were sequenced using automated DNA sequencers that worked on the principle of a method developed by Frederick Sanger.
(d) These sequences were then arranged based on some overlapping regions present in them.
Salient Features of Human Genome
(a) The human genome contains 3164.7 million nucleotide bases.
(b) The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million bases.
(c) Less than 2 per cent of the genome codes for proteins.
(d) Repeated sequences make up very large portion of the human genome.
(e) Repetitive sequences are stretches of DNA sequences that are repeated many times, sometimes hundred to thousand times.
(f) Chromosome 1 has most genes (2968), and the Y has the fewest (231).
(g) Scientists have identified about 1.4 million locations where single base DNA differences (SNPs - single Nucleotide Polymorphism) occur in humans.