The techniques of DNA diagnosis have found application in a quite different areas. This is important in areas as diverse as identifying cell culture, determining family relationships, in studies of animal behaviour, immigration problem, to identify criminals or murderer, disputed paternity and in forensic medicine.
The most accurate method of identification technique based on recombinant technology is called DNA fingerprinting or DNA typing or DNA profiling.
Principle: DNA fingerprinting is based on sequence polymorphism that occurs in the human genome and the genome of every other organisms. The sequence of polymorphisms are slight sequence differences, usually single base pair changes, that occur from individuals to individuals once every few hundred base pairs on average. Each difference from the consensus human genome sequence is generally present in only a fraction of the human population but every individual has some of them. These polymorphic loci are called minisatellite or VNTR (variable number tandem repeat). Thus form a haplotype which shows Mendelian inheritance among their offsprings. This locus is made up of a variable number of identical sequences join together in tandem.
One family of minisatellites in the human genome share a common core sequence, the core is G-C rich sequence of 10-15 bp showing an asymmetry of purine/ pyrimidine distributed on the two strands. These repeats are written as (C-A)n (G-T)n occur in 100000 blocks in every genome and appear to be uniformly distributed throughout the genome (value of n varies between 4 and 40). The successful application of these (C-A)n. (G-T)n repeats has led to the use of a variety of other di, tri- and tetranucleotide sequences for mapping.
1. DNA of the sample is first isolated whose DNA fingerprinting has to be made. Usually in forensic case the DNA is prepared from dried blood stains, sperms in vaginal swabs that had been stored for as long as two years. Sufficient DNA can be isolated fro freshly pulled hair roots, Polymorphism in mitochondrial DNA and class II HLA gene DQ? have been analysed from the shed hairs of several months old containing less than 1 ng of DNA.
2. Insufficient quantities of intact DNA from forensic samples will always be a problem. PCR may have a great impact in this area. From a small quantity of DNA, PCR can produce a large amounts of DNA. This is used as probe. This probe is made radioactive.
3. The DNA from the individual whose DNA is to be compared with the forensic sample is isolated and is cut into fragments by restriction enzymes.
4. DNA fragments after digestion of DNA from the genome are first separated according to their size by agarose gel electrophoresis. These dsDNAs are denatured by soaking the gel in alkali to make is ssDNAs.
5. The DNA fragments are transferred to nitocellulose paper by the Southern blot technique. The paper is then immersed in a solution containing a radioactively labelled DNA probe. Fragments to which the probe. Fragments to which the probe hybridizes are revealed by autoradiography.
Detection of forensic problem: The power of DNA fingerprinting was demonstrated by Alec Jeffreys in 1985, when a man had been accused of two rape-murders committed three years apart and had made a confession. Eventually the real murderer was caught and DNA fingerprinting confirmed the identification.
DNA from a semen sample obtained from a rape and murderer victim was analyzed along the DNA samples from the victim and two suspects.
Each of the DNA samples was cleaved into fragments and separated by gel electrophoresis.
Radioactive DNA probes were used to identify a small subset of these fragments that contained sequences complementary to the probe. The sizes of the fragments identified varied from one individual to the next. The different patterns for the three individuals (victim and two suspects) tested. One rape suspect’s DNA exhibits a banding pattern identical to that of the semen sample taken from the victim. More than one probe may be used to make a positive identification.