4.10 DNA Fingerprinting:
Genes present on chromosomes are responsible for determining characters of the organism as well as for inheritance of characters. Due to recombination of paternal and maternal genes, we differ from our parents. Differences also arise due to infrequent mutations that occur during gamete formation (cell division).
Due to all these factors, every individual has its unique genetic make-up, which may be called its Fingerprint.
The technique developed to identify a person with the help of DNA restriction analysis, is known as DNA profiling or DNA fingerprinting.
The technique of finger printing was first given by British geneticist, Dr. Alec Jeffreys in 1984.
DNA fingerprinting technique is based on identification of nucleotide sequence present in this wonder molecule.
About 99.9% of nucleotide sequence in all persons, is same. Only some short sequences of nucleotides differ from person to person.
In the population, every person shows unusual sequences of 20-100 base pairs, which are repeated several times. They are termed as Variable Number of Tandem Repeats (VNTRs).
The length of the regions having VNTRs is different in each individual and hence is the key factor in DNA profiling. Steps involved in DNA finger printing are as follows:
1. Isolation of DNA:
The DNA must be recovered from the cells or tissues of the body (host). Only small amount of tissue like blood, hair roots, skin, etc. is required.
2. Restriction digestion:
The isolated DNA is treated with restriction enzymes. The restriction enzymes cut the DNA into small fragments having variable lengths.
This phenomenon is called Restriction Fragment Length Polymorphism (RFLP).
3. Gel electrophoresis:
The DNA samples are loaded for agarose gel electrophoresis under an electric influence. The DNA fragments, which are negatively charged move to the positive pole. The movement of these fragments depends on length of the fragments. This results in formation of bands. dsDNA splits into ssDNA by alkali treatment.
4. Southern blotting:
The separated DNA fragments are transferred to a nylon membrane or a nitrocellulose filter paper by placing it over the gel and soaking them with filter paper overnight.
5. Selection of DNA probe:
A known sequence of single- stranded DNA is prepared. It is called DNA Probe. DNA Probe is obtained from organisms or prepared by cDNA preparation method. The DNA probe is labelled with radioactive isotopes.
6. Hybridization:
Probe DNA is added to the nitrocellulose filter paper containing host DNA. The single-stranded DNA probe pairs with the complementary base sequence of the host DNA strand. As a result DNA-DNA hybrids are formed on the nitrocellulose filter paper. Remaining single stranded DNA probe fragments are washed off.
7. Photography:
The nitrocellulose filter paper is photographed on an X-ray film by autoradiography. The film is analysed to determine the presence of hybrid DNA.
Application of DNA fingerprinting
1. In forensic science, DNA finger printing is used to solve problems of rape and some complicated murder cases.
2. DNA finger printing is used to find out the biological father or mother or both, of the child, in case of disputed parentage.
3. DNA finger printing is used in pedigree analysis in cats, dogs, horses and humans.
