DNA profiling (also called DNA fingerprinting and genetic fingerprinting) is the process of determining an individual's deoxyribonucleic acid (DNA) 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 suspects' profiles to DNA evidence to assess the likelihood of their involvement in the crime. Modern DNA profiling techniques are highly reliable, despite the fact that they only provide a fallible probabilistic estimate of the match between a suspect and an incriminating sample. DNA profiling is also used in paternity testing,Petersen, K., J.. Handbook of Surveillance Technologies. 3rd ed. Boca Raton, FL. CRC Press, 2012. p815 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 (FSS), 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, Imperial Chemical Industries (ICI) 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.
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 (identical) twins. DNA profiling uses repetitive sequences that are highly variable, called variable number tandem repeats (VNTRs), in particular short tandem repeats (STRs), 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 (RFLP). 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 (also called phenol–chloroform 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.
Besides concentrating the DNA, extraction also separates the DNA from materials that would degrade the DNA such as nucleases.
RFLP analysis
RFLP is an application of the southern blot technique. The scientists chooses a mix of restriction enzymes, enzymes that "cut" the short and specific sequences throughout the sample. The enzyme is used to digest the DNA sample. Gel electrophoresis of nucleic acids, which uses an electric field to cause different sizes of DNA to move at different speeds, is used to perform an initial separation by size.
At this point the DNA has been separated by size but remains invisible. A thin nitrocellulose or nylon membrane is pressed onto the gel for some time so that the DNA is imprinted as a pattern on the membrane. The membrane is then baked or exposed to ultraviolet light to permanently attach the DNA onto the membrane. To this membrane with baked-on DNA, a hybridization probe is used to highlight the part of the DNA that matches the sequence of the probe. The probe is usually either radioactive or chemiluminescent so that its location can be recorded as a picture of the "bands", each corresponding to a size-group of DNA that match the probe.
RFLP analysis works by comparing the position of bands (which corresponds to the sizes of probe-matching fragments) across different people.
Polymerase chain reaction (PCR)
PCR is a way to cyclically amplify portions of DNAs that match certain primer(s). It 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. Amplification is achieved by cycling the following three steps:
Denaturation: Heat mixture containing DNA to 95 °C so the two strands become separate from each other. Annealing: Cool the mixture to 50–65 °C so the primers can attach to the DNA by forming complementary base pairs. Extension: A DNA polymerase starts from where the primer binds to the DNA (duplex) and builds out a complementary chain of DNA.
By using either specific primers that target a certain area or random primers, PCR can be used for a great variety of analysis tasks.
Paired primers One classical use of PCR is to selective amplify an area between a pair of two primers; because the target fragment size would be known beforehand, no restriction enzymes are needed (though they certainly can be incorporated). The product can then be separated by size using gel electrophoresis, like described above for southern blotting, and visualized by transferring onto a membrane in the same way. The resulting image would show bands of expected size if the area exists, no band if nothing of appropriate size matches the area, or a different size if something matches but is a different length; all of these are good information for differentiating species of organisms.
If one targets area that is known to either match with a fixed size or not match in the overwhelming majority of cases, then the band either exists or doesn't. In this case, many pairs of primers that matches areas of different lengths can be used together (if they do not end up matching extra areas), allowing the presence or absence of them to be analyzed in a single PCR reaction. This is PCR fingerprinting.
STR analysis
A kind of human genetic variation that is easy to spot as size difference are the short tandem repeats (STR)s, where a very short and simple sequence is repeated many times. These kind of sites generate a lot of variation between people because they are "slippery": the DNA polymerase can slip and end up skipping or adding a few copies. STR analysis is a specific version of the classical paired-primer PCR fingerprinting, in that the primers are targeting areas with known STR. By targeting an area that varies greatly between people, it turns PCR fingerprinting from a species-identification technique used in biology into a family-, even individual-identifying technique usable in forensics.
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 (having a certain number of repeats at one locus does not change the likelihood of having any number of repeats at any other locus), 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 (1 × 1018) or more. However, DNA database searches showed much more frequent than expected false DNA profile matches.
Y-chromosome Standard STR analysis kits mostly focus on autosomal STRs, but most usually include one that tests for AMELY, the Y-chromosomal copy of the amelogenin gene, to indicate whether the Y chromosome is present. (This single data point could rule out 50% of suspects.) A very small amount of people have a variant of AMELY undetectable by this area, so some kits also add up to 4 Y-chromosomal indels and STRs.
Y chromosomal STRs are used in much of the same way as the autosomal STRs, with the added knowledge that it only passes down the paternal line. Kits specialized for Y STR test for up to 27 loci; 8 is the "minimal haplotype" set. The "Y haplotype reference database (YHRD)" has been created in 2000 as an online resource. It currently comprises more than 300,000 minimal (8 locus) haplotypes from world-wide populations as well as data on their frequency.
Sequencing