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Nature, Purpose & Scope
DNA or Deoxyribo nucleic acid is the genetic material present in the nucleus of cells in all living organisms including plants, animals, humans, etc. and DNA is unique for each and every human being just like finger prints. It is the DNA present in every one of us that makes us forensically distinguishable from each other. Hence, DNA is an excellent parameter for identifying an individual.
DNA profiling technique was developed in 1984 by English geneticist Alec Jeffreys of the University of Leicester in the course of a molecular research stud , and was first used to convict Colin Pitchfork in 1988 in the Enderby murders case in Leicestershire, England and was proven as an infallible method.
Transfer of DNA from one individual to other individual
The direct transfer of DNA from one individual to other individual or to an object can be used to link a suspect to a crime scene. This direct transfer could involve:
The suspect’s DNA deposited on the victim’s body or clothing;
The suspect’s DNA deposited on a object;
The suspect’s DNA deposited at a location;
The victim’s DNA deposited on the suspect’s body or clothing;
The victim’s DNA deposited on an object;
The victim’s DNA deposited at a location;
The witness’s DNA deposited on victim or suspect; or
The witness’s DNA deposited on an object or at a location
Current advances allow DNA Profiling (fingerprinting) to be carried out on forensic samples of human origin such as blood, blood stains, semen, seminal stains, vaginal swabs, tissues, bones, hair, teeth, saliva and other skeletal remains. Applying this technique, it is now possible to underrtake paternity/maternity testing for solving disputed parentage, swapping of child from hospitals, illegal adoption and immigration cases. It is possible to undertake examination of samples of animal or plant origin wherever required in cases of wildlife forensics, forensic botany, etc. DNA can be obtained from the following physical evidence that may be found at the crime scene:
Debris from fingernails or broken fingernails
Postage stamps (licked)
Envelope sealing flaps
Personal items : razor blade, chewing gum, wrist watch, ear wax, toothbrush
Tools handled by the suspect,
Any other item with which victim / suspect interac
Basic Concepts of DNA
Deoxyribonucleic acid, or DNA, is sometimes referred to as genetic blueprint because it stores the information necessary for passing down genetic attributes to future generations. Residing in every cell of our body (with the exception of red blood cells, which lack nuclei), DNA provides a ‘computer program’ that determines our physical features and many other attributes. The complete set of instructions for making a organism, i.e. all the DNA in a cell, is referred to collectively as its genome.
Half of the DNA is inherited from a person’s mother and the other half from his/her father. Siblings inherit different combinations of DNA from the same parents and are therefore different from each other. Each generation of people is a new and different combination of generic material from the previous generation. Except for identical twins, each person’s DNA is unique, although the technology available does not yet allow the examination of every single difference between people’s DNA.
Within human cells, DNA found in the nucleus of the cell (nuclear DNA) is divided into chromosomes. The human genome consists of 22 matched pairs of autosomal chromosomes and two sex determining chromosomes. Thus, normal human cells contain 46 different chromosomes or 23 pairs of chromosomes. Males are designated XY because they contain one copy of the X chromosome and one copy of the Y chromosome, while females contain two copies of the X chromosome and are designated XX.
Figure showing the double helix of the DNA
Further it has been scientifically proven that, except for mono-zygotic or identical twins, each individual can be differentiated from another by means of DNA. Composition and structure of DNA is the same in each and every human cell but differs from individual to individual. Given this uniqueness of DNA, physical evidence collected from crime scene can either link a suspect to the crime scene or eliminate him just like fingerprints.
Practical Applications of DNA Fingerprinting
1. Paternity and Maternity Because a person inherits his or her VNTRs from his or her parents, VNTR patterns can be used to establish paternity and maternity. The patterns are so specific that a parental VNTR pattern can be reconstructed even if only the children's VNTR patterns are known (the more children produced, the more reliable the reconstruction). Parent-child VNTR pattern analysis has been used to solve standard father-identification cases as well as more complicated cases of confirming legal nationality and, in instances of adoption, biological parenthood.
2. Criminal Identification and Forensics DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to determine guilt or innocence. VNTR patterns are also useful in establishing the identity of a homicide victim, either from DNA found as evidence or from the body itself.
3. Personal Identification The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen anytime in the foreseeable future. The technology required to isolate, keep on file, and then analyze millions of very specified VNTR patterns is both expensive and impractical. Social security numbers, picture ID, and other more mundane methods are much more likely to remain the prevalent ways to establish personal identification.
4. Diagnosis of Inherited Disorders
DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies in hospitals around the world. These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others.
Early detection of such disorders enables the medical staff to prepare themselves and the parents for proper treatment of the child. In some programs, genetic counselors use DNA fingerprint information to help prospective parents understand the risk of having an affected child. In other programs, prospective parents use DNA fingerprint information in their decisions concerning affected pregnancies.
5. Developing Cures for Inherited Disorders
Research programs to locate inherited disorders on the chromosomes depend on the information contained in DNA fingerprints. By studying the DNA fingerprints of relatives who have a history of some particular disorder, or by comparing large groups of people with and without the disorder, it is possible to identify DNA patterns associated with the disease in question. This work is a necessary first step in designing an eventual genetic cure for these disorders.
A marker is a piece of the DNA molecule that is associated with a certain trait of an organism. Markers help scientists determine the location (map) of genes that control important traits.The DNA fingerprinting technique is used with traditional genetic analysis of family pedigrees and breeding data to identify DNA markers that are inherited along with important traits. This process is sometimes referred to as marker-assisted selection.In marker-assisted selection, a scientist who knows which piece of DNA is associated with a desirable trait can select plants or animals that have that piece of DNA. It often is easier and less expensive to select plants or animals that have the DNA marker than it is to grow them to maturity and see if they develop the desirable trait.
A breeder traditionally uses the appearance (phenotype) of an animal or plant to judge the genotype (DNA) that has been inherited. However, there are problems when the phenotype is used to select individuals for breeding where all genes are difficult to observe or measure. Or homozygous individuals may be impossible to distinguish from heterozygous individuals, because the dominant A gene would cause them to look alike.
DNA fingerprinting can make the selection process more precise. If DNA markers are known for the genes, then DNA fingerprinting can accurately measure the genotype to ensure that the breeder correctly identifies homozygous and heterozygous individuals.DNA fingerprinting can also speed the selection process and reduce the costs considerably. DNA tests can be done on embryonic tissue. Even at this early stage, the DNA test accurately predicts the gene an individual possesses.
Before transgenic plants or animals can be developed, the DNA code necessary for the desired trait must be known and identified. Gene mapping, described earlier, is a method to identify the genes responsible for specific traits.
9. Legal Protection
Because of the uniqueness of DNA fingerprint data, the technique can be used to legally protect new varieties of plants or animals, whether they were developed by genetic engineering, tissue culture, or traditional methods. Using DNA fingerprints to identify and protect commercial varieties of crops or livestock is a relatively new application of DNA fingerprint technology.
History of DNA Profling
Technologies used for performing forensic DNA analysis differ in their ability to differentiate two individuals and in the speed with which results can be obtained. The speed of analysis has dramatically improved for forensic DNA analysis. DNA testing that previously took 6 or 8 weeks can now be performed in a few hours.
The human identity testing community has used a variety of techniques including single-locus probe and multi-locus probe RFLP methods and more recently PCR (polymerase chain reaction)-based assays. Numerous advances have been made in the last 15 years in terms of sample processing speed and sensitivity. Instead of requiring large blood stains with well-preserved DNA, minute amounts of sample, as little as a single cell in some cases, can yield a useful DNA profile.
Multi-locus RFLP probes are highly variable between individuals but require a great deal of labor, time, and expertise to produce a DNA profile. Analysis of multi-locus probes (MLP) cannot be easily automated, a fact that makes them undesirable as the demand for processing large numbers of DNA samples has increased. Deciphering sample mixtures, which are common in forensic cases, is also a challenge with MLP RFLP methods, which is the primary reason that laboratories went to single-locus RFLP probes used in serial fashion.
The best solution including a high power of discrimination and a rapid analysis speed has been achieved with short tandem repeat (STR) DNA markers. Also because STRs by definition are short, they can be analyzed three or more at a time. Multiple STRs can be examined in the same DNA test, or ‘multiplexed.’ multiplex STRs are valuable because they can produce highly discriminating results and can successfully measure sample mixtures and biological materials containing degraded DNA molecules. In addition, the detection of multiplex STRs can be automated, which is an important benefit as demand for DNA testing increases.
Mitochondrial DNA (mtDNA), with the lowest power of discrimination and longest sample processing time, can be very helpful in forensic cases involving severely degraded DNA samples or when associating maternally related individuals.
Over the past 15 years, there has been a gradual evolution in adoption of the various DNA typing technologies. when early methods for DNA analysis are superseded by new technologies, there is usually some overlap as forensic laboratories implement the new technology. Validation of the new methods is crucial to maintaining high-quality results.
DNA Technologies used in Forensic Investigations
Restriction Fragment Length Polymorphism (RFLP)
RFLP is a technique for analyzing the variable lengths of DNA fragments that result from digesting a DNA sample with a special kind of enzyme. This enzyme, a restriction endonuclease, cuts DNA at a specific sequence pattern know as a restriction endonuclease recognition site. The presence or absence of certain recognition sites in a DNA sample generates variable lengths of DNA fragments. They are then hybridized with DNA probes that bind to a complementary DNA sequence in the sample. The distance between the locations cut by restriction enzymes (the restriction sites) varies between individuals: so the length of the fragments varies, and the position of certain gel bands differs between individuals (thus polymorphism). This can be used to genetically tell individuals apart.RFLP is one of the original applications of DNA analysis to forensic investigation. With the development of newer, more efficient DNA-analysis techniques, RFLP is not used as much as it once was because it requires relatively large amounts of DNA. In addition, samples degraded by environmental factors, such as dirt or mold, do not work well with RFLP.
When DNA fingerprinting first began, RFLP analysis was used, though it has been almost completely replaced with newer techniques. RFLP analysis is performed by using a restriction enzyme to cut the DNA into fragments which are separated into bands during agarose gel electrophoresis. Next, the bands of DNA are transferred via a technique called Southern blotting from the agarose gel to a nylon membrane. This is treated with a radioactively-labelled DNA probe which binds to certain specific DNA sequences on the membrane. The excess DNA probe is then washed off. An X-ray film placed next to the nylon membrane detects the radioactive pattern. This film is then developed to make a visible pattern of bands called a DNA fingerprint. By using multiple probes targeting various polymorphisms in successive X-ray images, a fairly high degree of discrimination was possible. The primary drawback of RFLP is that the exact sizes of the bands are unknown and comparison to a molecular weight ladder is done in a purely qualitative manner. RFLP was a very time consuming method which required relatively high quantity of good quality DNA to be used (such as a dime sized blood drop). This made typing degraded samples such as those from evidence that had been exposed to the elements fairly difficult.
PCR (polymerase chain reaction) is used to make millions of exact copies of DNA from a biological sample. DNA amplification with PCR allows DNA analysis on biological samples as small as a few skin cells. With RFLP, DNA samples would have to be about the size of a quarter. The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. Great care, however, must be taken to prevent contamination with other biological materials during the identifying, collecting, and preserving of a sample.
With the invention of polymerase chain reaction, DNA fingerprinting took huge strides forward in both discriminating power and ability to recover information from very small starting samples. Commercial kits that used single nucleotide polymorphisms (SNPs) for discrimination became available. These kits use PCR to amplify specific regions with known variations and hybridize them to probes anchored on cards, which results in a colored spot corresponding to the particular sequence variation.
Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. CODIS is a software program that operates local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and missing persons. The odds that two individuals will have the same 13-loci DNA profile are about one in one billion.
The most prevalent method of DNA fingerprinting used today is based on PCR and uses STR. This method uses highly polymorphic regions that have short repeated sequences of DNA. Because different people have different numbers of repeat units, these regions of DNA can be used to discriminate between individuals. These STR loci (locations) are targeted with sequence-specific primers and are amplified using PCR. The DNA fragments that result are then separated and detected using electrophoresis. There are two common methods of separation and detection, capillary electrophoresis and gel electrophoresis.
The polymorphisms displayed at each STR region are by themselves very common, typically each polymorphism will be shared by around 5 - 20% of individuals. When looking at multiple loci, it is the unique combinations of these polymorphisms to an individual that makes this method discriminating as an identification tool. The more STR regions that are tested in an individual the more discriminating the test becomes.
Mitochondrial DNA Analysis
Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR. Nuclear DNA must be extracted from samples for use in RFLP, PCR, and STR; however, mtDNA analysis uses DNA extracted from another cellular organelle called a mitochondrion. While older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP and also for highly degraded samples, it is sometimes impossible to get a complete profile of the 13 CODIS STRs. These can be analyzed with mtDNA. In the investigation of cases that have gone unsolved for many years, mtDNA is extremely valuable as many copies of mtDNA can be found in a cell, while there may only be 1-2 copies of the nuclear DNA.
All mothers have the same mitochondrial DNA as their daughters. This is because the mitochondrion of each new embryo comes from the mother's egg cell. The father's sperm contributes only nuclear DNA. Comparing the mtDNA profile of unidentified remains with the profile of a potential maternal relative can be an important technique in missing person investigations. A difference of two more nucleotides is generally considered to be an exclusion.
The Y chromosome is passed directly from father to son i.e., it is paternally inherited, so the analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males or for analyzing biological evidence involving multiple male contributors. In other words, for identification of paternally related males.
Recent innovations have included the creation of primers targeting polymorphic regions on the Y-chromosome (Y-STR), which allows resolution of multiple male profiles, or cases in which a differential extraction is not possible.
DNA and Forensic Science
DNA evidence collection from a crime scene must be performed carefully and a chain of custody established in order to produce DNA profiles that are meaningful and legally accepted in court. DNA testing techniques have become so sensitive that biological evidence too small to be easily seen with the naked eye can be used to link suspects to crime scenes. The evidence must be carefully collected, preserved, stored, and transported prior to any analysis conducted in the laboratory.
It must be remembered that these tests are not always foolproof and should be used in conjunction with other evidence where possible. DNA fingerprinting has nevertheless affected the outcome of criminal investigations in a revolutionary way.
Any chromosome that is not a sex chromosome is called autosomal
Zygote is the cell formed by the union of a male sex cell (a sperm) and a female sex cell (an ovum or egg). The zygote develops into the embryo following the instruction in its genetic material, the DNA. Identical twins develop from one zygote that splits and forms two embryos, thus they share the same DNA.
13-loci DNA profile are about one in one billion.
The most prevalent method of DNA fingerprinting used to