DNA profiling
DNA profiling is the process of determining an individual's deoxyribonucleic acid characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding.
DNA profiling is a forensic technique in criminal investigations, comparing criminal suspects' profiles to DNA evidence so as to assess the likelihood of their involvement in the crime. It is also used in paternity testing, to establish immigration eligibility, and in genealogical and medical research. DNA profiling has also been used in the study of animal and plant populations in the fields of zoology, botany, and agriculture. DNA profiling was discovered by British geneticist Sir Alec Jeffreys in 1984 while he was working at the University of Leicester. He developed the technique of genetic fingerprinting by realizing that some regions of DNA have highly variable repetitive sequences that are unique to each individual.
Background
Starting in the mid 1970s, scientific advances allowed the use of DNA as a material for the identification of an individual. The first patent covering the direct use of DNA variation for forensics was issued in 1997, continued from an application first filed by Jeffrey Glassberg in 1983, based upon work he had done while at Rockefeller University in the United States in 1981.British geneticist Sir Alec Jeffreys independently developed a process for DNA profiling in 1984 while working in the Department of Genetics at the University of Leicester. Jeffreys discovered that a DNA examiner could establish patterns in unknown DNA. These patterns were a part of inherited traits that could be used to advance the field of relationship analysis. These discoveries led to the first use of DNA profiling in a criminal case.
The process, developed by Jeffreys in conjunction with Peter Gill and Dave Werrett of the Forensic Science Service, was first used forensically in the solving of the murder of two teenagers who had been raped and murdered in Narborough, Leicestershire in 1983 and 1986. In the murder inquiry, led by Detective David Baker, the DNA contained within blood samples obtained voluntarily from around 5,000 local men who willingly assisted Leicestershire Constabulary with the investigation, resulted in the exoneration of Richard Buckland, an initial suspect who had confessed to one of the crimes, and the subsequent conviction of Colin Pitchfork on January 2, 1988. Pitchfork, a local bakery employee, had coerced his coworker Ian Kelly to stand in for him when providing a blood sample—Kelly then used a forged passport to impersonate Pitchfork. Another coworker reported the deception to the police. Pitchfork was arrested, and his blood was sent to Jeffreys' lab for processing and profile development. Pitchfork's profile matched that of DNA left by the murderer which confirmed Pitchfork's presence at both crime scenes; he pleaded guilty to both murders. After some years, a chemical company named Imperial Chemical Industries introduced the first ever commercially available kit to the world. Despite being a relatively recent field, it had a significant global influence on both criminal justice system and society.
Image:D1S80Demo.png|thumb|Variations of VNTR allele lengths in 6 individuals
Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different that it is possible to distinguish one individual from another, unless they are monozygotic twins. DNA profiling uses repetitive sequences that are highly variable, called variable number tandem repeats, in particular short tandem repeats, also known as microsatellites, and minisatellites. VNTR loci are similar between closely related individuals, but are so variable that unrelated individuals are unlikely to have the same VNTRs.
Before VNTRs and STRs, people like Jeffreys used a process called restriction fragment length polymorphism. This process regularly used large portions of DNA to analyze the differences between two DNA samples. RFLP was among the first technologies used in DNA profiling and analysis. However, as technology has evolved, new technologies, like STR, emerged and took the place of older technology like RFLP.
The admissibility of DNA evidence in courts was disputed in the United States in the 1980s and 1990s, but has since become more universally accepted due to improved techniques.
Profiling processes
DNA extraction
When a sample such as blood or saliva is obtained, the DNA is only a small part of what is present in the sample. Before the DNA can be analyzed, it must be extracted from the cells and purified. There are many ways this can be accomplished, but all methods follow the same basic procedure. The cell and nuclear membranes need to be broken up to allow the DNA to be free in solution. Once the DNA is free, it can be separated from all other cellular components. After the DNA has been separated in solution, the remaining cellular debris can then be removed from the solution and discarded, leaving only DNA. The most common methods of DNA extraction include organic extraction, Chelex extraction, and solid-phase extraction. Differential extraction is a modified version of extraction in which DNA from two different types of cells can be separated from each other before being purified from the solution. Each method of extraction works well in the laboratory, but analysts typically select their preferred method based on factors such as the cost, the time involved, the quantity of DNA yielded, and the quality of DNA yielded.RFLP analysis
RFLP stands for restriction fragment length polymorphism and, in terms of DNA analysis, describes a DNA testing method which utilizes restriction enzymes to "cut" the DNA at short and specific sequences throughout the sample. To start off processing in the laboratory, the sample has to first go through an extraction protocol, which may vary depending on the sample type or laboratory SOPs. Once the DNA has been "extracted" from the cells within the sample and separated away from extraneous cellular materials and any nucleases that would degrade the DNA, the sample can then be introduced to the desired restriction enzymes to be cut up into discernable fragments. Following the enzyme digestion, a Southern Blot is performed. Southern Blots are a size-based separation method that are performed on a gel with either radioactive or chemiluminescent probes. RFLP could be conducted with single-locus or multi-locus probes. Incorporating the multi-locus probes allowed for higher discrimination power for the analysis, however completion of this process could take several days to a week for one sample due to the extreme amount of time required by each step required for visualization of the probes.Polymerase chain reaction (PCR) analysis
This technique was developed in 1983 by Kary Mullis. PCR is now a common and important technique used in medical and biological research labs for a variety of applications.PCR, or Polymerase Chain Reaction, is a widely used molecular biology technique to amplify a specific DNA sequence.
Amplification is achieved by a series of three steps:
1- Denaturation : In this step, the DNA is heated to 95 °C to dissociate the hydrogen bonds between the complementary base pairs of the double-stranded DNA.
2-Annealing : During this stage the reaction is cooled to 50-65 °C. This enables the primers to attach to a specific location on the single -stranded template DNA by way of hydrogen bonding.
3-Extension : A thermostable DNA polymerase which is Taq polymerase is commonly used at this step. This is done at a temperature of 72 °C. DNA polymerase adds nucleotides in the 5'-3' direction and synthesizes the complementary strand of the DNA template.
STR analysis
The system of DNA profiling used today is based on polymerase chain reaction and uses simple sequences.From country to country, different STR-based DNA-profiling systems are in use. In North America, systems that amplify the CODIS 20 core loci are almost universal, whereas in the United Kingdom the DNA-17 loci system is in use, and Australia uses 18 core markers.
The true power of STR analysis is in its statistical power of discrimination. Because the 20 loci that are currently used for discrimination in CODIS are independently assorted, the product rule for probabilities can be applied. This means that, if someone has the DNA type of ABC, where the three loci were independent, then the probability of that individual having that DNA type is the probability of having type A times the probability of having type B times the probability of having type C. This has resulted in the ability to generate match probabilities of 1 in a quintillion or more. However, DNA database searches showed much more frequent than expected false DNA profile matches.