The introduction of DNA technology in forensic science is nearly two decades old. During this time, identification based on differences that exist in DNA molecule has moved from an experimental technique to an established crime-solving too! For police and prosecutors throughout the world. Never before have had prosecutors had such a powerful tool at their disposal both to convict the guilty and exonerate the wrongly accused. The key insight came from the discovery of a technique called DNA Fingerprinting, first described by a British scientist Sir Alec Jeffrys in 1985. A DNA fingerprint can simply be called the genetic photograph of an individual. Like fingerprints that came into use by detectives and police labs in 1930s, each individual has a unique DNA fingerprint. A conventional fingerprint can be changed by plastic surgery, but a DNA fingerprint cannot be altered by any known treatment because, DNA is a component of virtually every cell in the human body.
Thus, by using modern molecular biological techniques it is now possible to generate a DNA fingerprint from any trace of biological specimen like blood, hair, saliva, semen, skin, tissue or bone in the laboratory within 24 hours of time. Since its discovery, DNA fingerprinting of biological material has become one of the most powerful tools for personal identification in forensic medicine and criminal investigation. First used in forensic casework in 1987 in UK to convict a criminal who raped and murdered two teenage school girls. More than 100 countries in the world have already introduced the technology in their legal system.
What is DNA?
DNA or Deoxyribo Nucleic Acid is the fundamental building block for an individual's entire genetic makeup. It is the hereditary blueprint passed on to us by our parents. All the characteristics of a living organism, including humans is essentially determined by the information they contain in their DNA molecule. Humans are multi-cellular organism and are composed of approximately 100 trillion cells. In the inner part of the cell lies the nucleus which, in turn contain chromosomes. Chromosomes are made of DNA. The molecular structure of DNA can be imagined as a twisted ladder. Each strand of the ladder is a long polymer consisting of four different subunits or bases. These bases are adenine, guanine, thymine and cytosine and are represented by four letters (A, G, T and C). The information contained in DNA is the sequence of these four letter along the strand and represent the genetic alphabet. For example, the sequence ACGCT represent different information than the sequence AGTCC, the same way, the word STAR has a different meaning than ARTS or RATS even though they use the same letters.
What is DNA Fingerprinting?
DNA fingerprinting which is now most popularly known as DNA profiling, is a method of isolating and making images of DNA sequences. Each human cell (except reproductive cells) contains approximately 3 billion base pairs of DNA. Among these about 10% of the DNA encodes over 40,000 genes. A gene is a segment of DNA that is responsible for a particular characteristic such as. Hair color, eye color, stature, bone density, habits, likes, dislikes etc. Genes are separated from each other by non-coding DNAs, called intrones and constitute about 90% of the total DNA. lntrones or non-coding DNAs have no known function and therefore sometimes called junk DNA. In humans about 99.9% of all 3 billion nucleotides we inherit from our parents are identical among all individuals and difference exists in only 0.1% DNA. Interestingly. This 0.1% DNA lies in these non-coding regions. Within these regions it has been found that certain DNA sequences are repeated over and over again and the number of repeated sections could differ from individual to individual. Such variable stretches are termed as polymorphic (meaning many forms). One such form of variable sequence is STR (Short Tandem Repeats) or microsatellite sequences. These sequences are usually 2-7 base pair long and repeated between 7 to 40 times. Several hundred STR sequences are spread over the entire human genome and situated exclusively in the non-coding regions. DNA fingerprinting is thus a quick way of identifying individuals by comparing sequences of these regions. It does not involve the analysis of the whole genome, rather a snapshot of the polymorphic regions.
Technological basis of DNA profiling
Forensic DNA typing usually consists of comparing DNA evidence (i.e DNA extracted from a biological sample left at a crime scene) with suspect DNA (i.e. DNA extracted from the blood of a suspect). The two methods for DNA fingerprinting eventually found their way to courtroom are: RFLP (Restriction Fragment Length Polymorphism) and PCR (Polymerase Chain Reaction). Though RFLP was the initial method adopted for use, it is no longer the preferred approach in majority of the forensic laboratories because, it requires good amount of non degraded DNA which is often very difficult to obtain from the crime scene. Moreover, it requires radioactive labeling and may take 1-2 weeks for the result to be processed. The technology most widely used now-a-days is PCR technology.
PCR is a technique that allows someone to amplify or copy a specific DNA sequence from trace amount of DNA. PCR based DNA profiling employs the amplification of several microsatellite or STR regions in a single reaction. Since STRs are smaller sequences (2-7 base pair long), they are more likely to survive degradation and analysis can be done on less than one billionth of a gram (a nanogram) of DNA within 24 hours. There are more than 100 STR regions or loci spread over the entire human genome. In practice, DNA is first isolated from biological specimen (blood, hair, skin, semen, etc) and 10-15 STR loci are amplified using specific primers (small DNA fragments of - 25-30 base pair) that flank on either side of the sequence of interest. The PCR products are then separated by capillary electrophoresis (a technique that separates DNA molecule on the basis of size) using a Genetic Analyzer. Since these primers are labeled with fluorescent dye, after electrophoresis the PCR products are detected by a laser beam embedded in the analyzer. The fluorescent signal that is emitted from each of the amplicon is detected as individual peaks. These data are then analyzed by Genemapper ID-X software and assigned numerically (digits 1, 2, 3 etc). In humans, the chromosomes occur as homologous pair, this means, we have two copies of every chromosome. So, a person may be homozygous (same number of repeats) or heterozygous (different number of repeats) at each STR site. The figure above shows a DNA profile that has been generated using 15 STR loci. At D7S820, CSF1P0, TH01 and TPDX loci the person is homozygous, on the other hand, at rest of the loci the person is heterozygous (e.g. different number of repeats from homologous pairs of chromosomes). If the data are expressed digitally the DNA profile of that person can be written as above. When a DNA profile is generated in this using 15 STR loci, the probability of finding two individual with same DNA profile is 1 in a trillion, which is more than the world's population. Autosomal STR markers described above are used on a routine basis but, in certain circumstances Y-chromosome STR, X-chromosome or Mitochondrial DNA also provide valuable information.
Y-Chromosome STR: During the production of gametes (sperm or eggs), the paired chromosomes separate so that each gamete ends up with only one of each chromosome pair. However, before separation occurs, the paired autosomes exchange and reshuffle their DNA at every generation through recombination. In females this exchange process also takes place between the two X chromosomes but, in males unmatched X and chromosomes do not exchange DNA except at the end of the two chromosomes called pseudoautosomal regions. The pseudoautosomal region get their name because any genes located within them are inherited just like any autosomal genes.
The region located outside the pseudoautosomal regions is called non- pseudoautosomal or non-recombining portion of the Y chromosome (NRPY). Due to these unique properties, Y chromosome pass down from father to son largely unchanged. A man's Y chromosome thus represent a unique record of his paternal inheritance.
Y chromosome specific polymorphisms have proved to be especially useful in forensic casework. The application of Y chromosome polymorphism is most important in deficiency paternity testing when a male offspring is in question. They are also used for the analysis of male DNA fraction in stains involving male/female mixtures, the most common biological material available in sexual crimes. Y chromosome polymorphisms can also be used to reconstruct a history of human paternal lineage.
Mitochondrial DNA: Mitochondria is a sub-cellular organelle that works as a power house of the cell. Besides nuclear DNA, mitochondria contain a unique circular piece of DNA which is only 16,559 base pairs in length. 300,000 times less than the total DNA molecules present in the nucleus of a human cell. The mitochondrial genome contains a 1100 bp noncoding displacement loop (d-loop) or central region. The d-loop contains two hypervariable regions designated as HV1 and HV2. These hypervariable regions are the area containing most of the sequence variability and are the target areas for assessing sequence variability in mtDNA between individuals. One of the most important characteristic of mtDNA is that it follows a maternal inheritance pattern and inherited entirely from egg. It therefore can pass from generation to generation without any sexual recombination. The only change in mtDNA sequence may result from random mutation. Because of its
unique mode of transmission personal identification is also possible by comparing the mitochondrial DNA sequence with any maternally related individual. This of course requires the sequencing of HV1 or FIV2 region. Mitochondrial DNA IS useful when nuclear DNA analysis fails due to degradation and to establish maternal lineage.
X-chromosome (X-DNA) STR testing: An X-chromosome DNA test looks at markers on your X-chromosome(s). Males have one X-chromosome that they inherit exclusively from their mother, and females have two X-chromosomes that they inherit from both parents, one from their father and one from their mother. This creates a unique inheritance pattern that while challenging to follow may provide many insights into one’s maternal heritage. X-STRs have been proven to be useful in case of deficiency paternity testing and in effective mother–son kinship and father–daughter testing.
