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Human body is usually composed of an average six billion cells. DNA present in these cells has a large nuclear and a small mitochondrial component. The total DNA content in a single somatic cell is about 3 X 106. Mitochondrial DNA is circular and it constitutes about 5% of total DNA content. Out of total DNA, approximately 10% of the genome accounts for the estimated 50,000 to 1, 00,000 genes, which determine the character of an individual and carry it from one generation to next. Each gene has a definite sequence of base pairs. Rest of the DNA constitute about 90% of total genomic DNA is considered as "Junk", that it may have some as yet unknown functions, but it dose not code any known particular gene.
In 1985, Dr. Alec Jeffreys and colleagues at Leicester University, while studying the sequence of base pairs in myoglobin gene, observed that at certain areas DNA sequences are repeated over and over again in a chain like manner. They termed this phenomenon as "tandem repeat" and such repeat for short sequence were named as "minisatellite". The number of these repeat units is highly variable so that most people have different amount of these repeat units inherited from their mother and father. They are excellent sites to use in assessing differences among humans. The areas having such regions are termed as "Hyper Variable Regions" (HVRs). About 1500 HVRs are present in human genome. The variation pattern of such repetitive sequences of nucleotides in HVRs is so high that it is highly unlikely for the same combination to be repeated in DNA of another person. The pattern of HVRs in an individual are as unique as fingerprints. The technique which detects HVRs in DNA is known as "DNA Fingerprinting" or "Genetic Fingerprints". The probability of two random individuals sharing same DNA fingerprinting pattern is about 1 in 1023.
DNA Fingerprinting has found great importance in field of Forensic investigation. It has also a successful application in setting of immigration and also in wild life conservation.
Techniques of DNA Fingerprinting:
There are mainly two approaches to determine DNA profiling pattern. First is by using restriction enzyme to cut the DNA in to pieces and separate the pieces using electrophoresis. The second way is the use of PCR process to copy or amplify the tandem repeat areas and then separate them by electrophoresis.
I. Restriction Fragment Length Polymorphism (RFLP) :
The technique is based on determination of the length of repeated sequences in specified areas of an individual DNA. The repeat sequences are cut from DNA with the help of an enzyme termed as "Restriction Endonuclease". These enzymes cut through both strands of DNA only at certain recognition sites. The fragments are then separated in electric field according to their sizes by the technique known as electrophoresis. Now the separated fragments are transferred from gel to nylon membrane by a technique known as "Southern Blotting Technique". After transfer to nylon membrane, locations of repeat sequences are to be established. This is accomplished by using DNA probes. The DNA probes are short sequences that are complementary to the repeat sequences in VNTR (Variable Number of Tandem Repeat) regions of DNA. The DNA Probes are labeled with radioactive isotope (P32). The labeled probes are hybridized to the nylon membrane. The membrane is then placed in between two X-ray films and the emission of radioactive particles from probe produces a pattern in X-ray film. The band like pattern obtained by this technique is specific to each individual. Chemiluminescent tagged probes also work in a similar manner.
The probes used in above techniques are of two categories. The first category of probe detects tandem repeats at several Hyper Variable loci and produce extremely complex pattern consisting of several bands. These probes are known as 'Multi Locus Probes' (MLPs). The probability of identical patterns in any two randomly selected individuals in the population obtained by MLPs is about 1: 1013 to 1:1023. The multi locus band patterns are known as 'DNA Fingerprinting' because of their capability to individualize the person.The second category of probes detect a single Hyper Variable locus, is known as 'Single Locus Probes' (SLPs).These probes produces one or two band pattern . A set of 4 to 5 SLPs are required to obtain meaningful individual specific pattern.
Polymerase Chain Reaction (PCR):
The Polymerase Chain Reaction is in vitro DNA amplification process that can amplify specific segments of DNA by as much as 108 folds. PCR reaction requires two oligo nucleotide primers designed to hybridize opposite strands of the target sequence and DNA polymerase (Taq polymarese) enzyme. The PCR reaction involves repeated cycles of heat suitable for denaturation of DNA, annealing of the primers to their complementary sequences and extension of annealed primers with enzyme DNA polymerase. Thus DNA by this process synthesized across the region between the primers, effectively doubles the amount of DNA segment. Since the extension products are also complimentary to and capable of binding primers, successive cycles of amplification continue to double the amount of DNA synthesized than the previous cycle.
The PCR Technique is extremely useful for analysis of forensic samples, in which the evidence sample is very minute or degraded. The most popular PCR based techniques are as follows:
Allele specific oligonucleotide (ASO) amplification:
For allele specific oligo nucleotide (ASO) DNA amplification, the DNA to be identified must be a part of well characterized gene, which must be polymorphic in nature i.e. there must exist multiple forms (alleles) of the gene, that differ in their nucleotide sequence. HLADQA1 (Human Leucocytes Antigen) and poly markers (LDLR, GYPA, HGBB, D7S8 and GC) are important polymorphic genes. These markers can be analyzed by 'reverse dot-blot technique'. In this technique probes are immobilized on nylon membrane. The PCR amplified DNA fragments are then hybridized on the membrane containing immobilized oligonucleotide probes . The hybridized DNA is visualized with Biotin-Streptavidine method to obtain blue colored dots.
B .Amplified Fragment Length Polymorphism (AFLP):
At certain regions of HVRs short repetitive DNA sequences of 8 to 16 bp size and are arranged in tandem repeat manner, which can be amplified by PCR . The amplified product of such region containing polymorphism in their length (size) due to difference in number of tandem repeats. Greater number of repeats produces larger PCR product because more DNA is amplified in between the primers. The amplified DNA fragment can be separated by electrophoresis according to their sizes which can visualized by silver staining method.
The D1S80 locus is an example of AFLP which contains repetitive DNA sequence that are arranged as repeat units ( tandem ) are repeated 14 to over 41 times for each allele .
Short Tandem Repeats (STRs):
The most important example of AFLP is Short Tandem Repeats (STRs). These are tandemly repeat units of small size (2 to 7 bp ) and are highly informative. Alleles of these loci are differentiated by number of copies of repeat sequence contained within the amplified region. Several dozens of STR loci have been investigated for human identification testing. These STR loci are found on almost every chromosome and are well distributed in human genome. Tetra-nucleotide repeats are most popular among forensic scientists due to their fidelity in PCR amplification, although tri- and penta-nucleotide repeats are also in use. If multiple STR loci are examined, high degree of discrimination among individuals can be achieved. For this purpose generally 10 to 15 STR loci are amplified by PCR in a single reaction tube. The set of primers added in the reaction tube which is labeled with different fluorescent dyes on the 5'-end of a primer. The amplified products are run on Automated DNA sequencer system under suitable electrophoretic conditions. As the separated fluorescent tagged DNA fragments passes through a laser beam in the instrument, the fluorescent signals are scanned across the gel via laser-induced device and band pattern is of different STR loci are obtained.
In forensic investigation DNA profiling has been established as a powerful investigation tool. The discriminating power of DNA typing methods are million fold greater than conventional markers. The availability of DNA typing in all biological materials exposed under various environmental conditions have been well studied and documented. The DNA profile can obtained from all biological materials including blood, semen, saliva, muscle tissues, skeletal remains, hair roots, teeth etc. Owing to these tremendous potentials, DNA typing has become a powerful tool for investigation of serious criminal offences like homicide, rape, accidental cases, identification of victim from body remnants and also in paternity / maternity dispute cases.
The DNA profile obtained from evidence samples recovered from scene of offence can be compared with those of suspects DNA to make a valid conclusions. If it is different, the suspect is excluded as donor of the evidence material.
In paternity testing, DNA profile of mother and child are obtained to determine which half of child's DNA are inherited from mother, the other half is inherited from father. If a man does not have DNA types in his profile that matches with the paternal profile of the child, he is excluded as father.
In cases in which body fragments or highly decomposed body is recovered from scene of offence, the DNA profiles obtained from the same is compared with those of close relatives of the victim to obtain conclusive opinion about victim's identification.
Globally as well as in India DNA fingerprinting techniques are now widely accepted in courts and judgments are being given based on the conclusions obtained by this technology and has empowered low enforcement agencies.