DNA profiling (also called DNA testing, DNA typing, or genetic fingerprinting) is a technique employed by forensic scientists to assist in the identification of individuals on the basis of their respective DNA profiles.
Although the vast majority of human DNA sequences are common in humans, DNA profiling makes use of highly variable repeat sequences, called variable number tandem repeats (VNTR). These loci are highly similar between very closely related humans, but variable enough so that it is extremely unlikely for unrelated humans to have the same alleles.
DNA profiling commences with the extraction of an individual's DNA (typically called a "reference sample"). The most desirable method of collecting a reference sample is the use of a buccal swab, as this reduces the possibility of contamination. When this is not available (eg because a court order may be needed and not obtainable) other methods may need to be used to collect a sample of blood, saliva, semen, or other appropriate fluid or tissue from personal items (e.g. toothbrush, razor, etc) or from stored samples (e.g. banked sperm or biopsy tissue). Samples obtained from blood relatives (biological relative) can provide an indication of an individual's profile, as could human remains which had been previously profiled.
A reference sample is then analysed to create the individual's DNA profile using one of a number of techniques.
The first methods used for DNA profiling involved restriction enzyme digestion, followed by Southern blot analysis. Although polymorphisms can exist in the restriction enzyme cleavage sites, more commonly the enzymes and DNA probes were used to analyze VNTR loci. However, the Southern blot technique is laborious, and requires large amounts of undegraded sample DNA. Also, Jeffreys' original technique looked at many minisatellite loci at the same time, increasing the observed variability, but making it hard to discern individual alleles (and thereby precluding parental testing). These early techniques have been supplanted by PCR-based assays.
With the invention of the polymerase chain reaction (PCR) technique, DNA profiling took huge strides forward in both discriminating power and the ability to recover information from very small (or degraded) starting samples. PCR greatly amplifies the amounts of a specific region of DNA, using oligonucleotide primers and a thermostable DNA polymerase. Early assays such as the HLA-DQ alpha reverse dot blot strips grew to be very popular due to their ease of use, and the speed with which a result could be obtained. However they were not as discriminating as RFLP. It was also difficult to determine a DNA profile for mixed samples, such as a vaginal swab from a sexual assault victim.
Fortunately, the PCR method is readily adaptable for analyzing VNTR loci. In the United States the FBI has standardized a set of 13 VNTR assays for DNA typing, and has organized the CODIS database for forensic identification in criminal cases. Similar assays and databases have been set up in other countries. Also, commercial kits are available that analyze single nucleotide polymorphisms (SNPs). 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.
The most prevalent method of DNA profiling used today is based on PCR and uses short tandem repeats (STR). This method uses highly polymorphic regions that have short repeated sequences of DNA (the most common is 4 bases repeated, but there are other lengths in use, including 3 and 5 bases). Because different unrelated people have different numbers of repeat units, these regions of DNA can be used to discriminate between unrelated 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 (CE) 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 combination 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.
From country to country, different STR-based DNA-profiling systems are in use. In North America systems which amplify the CODIS 13 core loci are almost universal, while in the UK the SGM+ system, which is compatible with The National DNA Database in use. Whichever system is used, many of the STR regions under test are the same. These DNA-profiling systems are based around multiplex reactions, whereby many STR regions will be under test at the same time.
Capillary electrophoresis works by electrokinetically (movement through the application of an electric field) injecting the DNA fragments into a thin glass tube (the capillary) filled with polymer. The DNA is pulled through the tube by the application of an electric field, separating the fragments such that the smaller fragments travel faster through the capillary. The fragments are then detected using fluorescent dyes that were attached to the primers used in PCR. This allows multiple fragments to be amplified and run simultaneously, a procedure known as multiplexing. Sizes are assigned using labeled DNA size standards that are added to each sample, and the number of repeats are determined by comparing the size to an allelic ladder, a sample that contains all of the common possible repeat sizes. Although this method is expensive, larger capacity machines with higher throughput are being used to lower the cost/sample and reduce backlogs that exist in many government crime facilities.
Gel electrophoresis acts using similar principles as CE, but instead of using a capillary, a large polyacrylamide gel is used to separate the DNA fragments. An electric field is applied, as in CE, but instead of running all of the samples by a detector, the smallest fragments are run close to the bottom of the gel and the entire gel is scanned into a computer. This produces an image showing all of the bands corresponding to different repeat sizes and the allelic ladder. This approach does not require the use of size standards, since the allelic ladder is run alongside the samples and serves this purpose. Visualization can either be through the use of fluorescently tagged dyes in the primers or by silver staining the gel prior to scanning. Although it is cost-effective and can be rather high throughput, silver staining kits for STRs are being discontinued. In addition, many labs are phasing out gels in favor of CE as the cost of machines becomes more manageable.
The true power of STR analysis is in its statistical power of discrimination. In the US, there are 13 core loci (DNA locations) that are currently used for discrimination in CODIS. Because these loci are independently assorted (having a certain number of repeats at one locus doesn't 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, we can say that the probability of 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 with 18 zeros after it) or more. However, since there are about 12 millions monozygotic twins on Earth, that theoretical probablitity is useless. For example, the actual probability that 2 random persons have the same DNA is only 1 in 3 trillions.
Another technique, AmpFLP, or amplified fragment length polymorphism was also put into practice during the early 1990s. This technique was also faster than RFLP analysis and used PCR to amplify DNA samples. It relied on variable number tandem repeat (VNTR) polymorphisms to distinguish various alleles, which were separated on a polyacrylamide gel using an allelic ladder (as opposed to a molecular weight ladder). Bands could be visualized by silver staining the gel. One popular locus for fingerprinting was the D1S80 locus. As with all PCR based methods, highly degraded DNA or very small amounts of DNA may cause allelic dropout (causing a mistake in thinking a heterozygote is a homozygote) or other stochastic effects. In addition, because the analysis is done on a gel, very high number repeats may bunch together at the top of the gel, making it difficult to resolve. AmpFLP analysis can be highly automated, and allows for easy creation of phylogenetic trees based on comparing individual samples of DNA. Due to its relatively low cost and ease of set-up and operation, AmpFLP remains popular in lower income countries.
For highly degraded samples, it is sometimes impossible to get a complete profile of the 13 CODIS STRs. In these situations, mitochondrial DNA (mtDNA) is sometimes typed due to there being many copies of mtDNA in a cell, while there may only be 1-2 copies of the nuclear DNA. Forensic scientists amplify the HV1 and HV2 regions of the mtDNA, then sequence each region and compare single nucleotide differences to a reference. Because mtDNA is maternally inherited, directly linked maternal relatives can be used as match references, such as one's maternal grandmother's sister's son. A difference of two or more nucleotides is generally considered to be an exclusion. Heteroplasmy and poly-C differences may throw off straight sequence comparisons, so some expertise on the part of the analyst is required. mtDNA is useful in determining unclear identities, such as those of missing persons when a maternally linked relative can be found. mtDNA testing was used in determining that Anna Anderson was not the Russian princess she had claimed to be, Anastasia Romanov.
mtDNA can be obtained from such material as hair shafts and old bones/teeth.
The U.S. Patriot Act of the United States provides a means for the U.S. government to get DNA samples from other countries if they are either a division of, or head office of, a company operating in the U.S. Under the act, the American offices of the company can't divulge to their subsidiaries/offices in other countries the reasons that these DNA samples are sought or by whom.
When a match is made from a National DNA Databank to link a crime scene to an offender who has provided a DNA Sample to a databank that link is often referred to as a cold hit. A cold hit is of value in referring the police agency to a specific suspect but is of less evidential value than a DNA match made from outside the DNA Databank..
In the early days of the use of genetic fingerprinting as criminal evidence, juries were often swayed by spurious statistical arguments by defense lawyers along these lines: given a match that had a 1 in 5 million probability of occurring by chance, the lawyer would argue that this meant that in a country of say 60 million people there were 12 people who would also match the profile. This was then translated to a 1 in 12 chance of the suspect being the guilty one. This argument is not sound unless the suspect was drawn at random from the population of the country. In fact, a jury should consider how likely it is that an individual matching the genetic profile would also have been a suspect in the case for other reasons. Another spurious statistical argument is based on the false assumption that a 1 in 5 million probability of a match automatically translates into a 1 in 5 million probability of guilt and is known as the prosecutor's fallacy.
When using RFLP, the theoretical risk of a coincidental match is 1 in 100 billion (100,000,000,000), although the practical risk is actually 1 in 1000 because monozygotic twins are 0.2% of the human population. Moreover, the rate of laboratory error is almost certainly higher than this, and often actual laboratory procedures do not reflect the theory under which the coincidence probabilities were computed. For example, the coincidence probabilities may be calculated based on the probabilities that markers in two samples have bands in precisely the same location, but a laboratory worker may conclude that similar—but not precisely identical—band patterns result from identical genetic samples with some imperfection in the agarose gel. However, in this case, the laboratory worker increases the coincidence risk by expanding the criteria for declaring a match. Recent studies have quoted relatively high error rates which may be cause for concern In the early days of genetic fingerprinting, the necessary population data to accurately compute a match probability was sometimes unavailable. Between 1992 and 1996, arbitrary low ceilings were controversially put on match probabilities used in RFLP analysis rather than the higher theoretically computed ones Today, RFLP has become widely disused due to the advent of more discriminating, sensitive and easier technologies.
STRs do not suffer from such subjectivity and provide similar power of discrimination (1 in 10^13 for unrelated individuals if using a full SGM+ profile) It should be noted that figures of this magnitude are not considered to be statistically supportable by scientists in the UK, for unrelated individuals with full matching DNA profiles a match probability of 1 in a billion (one thousand million) is considered statistically supportable (Since 1998 the DNA profiling system supported by The National DNA Database in the UK is the SGM+ DNA profiling system which includes 10 STR regions and a sex indicating test. However, with any DNA technique, the cautious juror should not convict on genetic fingerprint evidence alone if other factors raise doubt. Contamination with other evidence (secondary transfer) is a key source of incorrect DNA profiles and raising doubts as to whether a sample has been adulterated is a favorite defense technique. More rarely, Chimerism is one such instance where the lack of a genetic match may unfairly exclude a suspect.
Members of the Jury, if you accept the scientific evidence called by the Crown, this indicates that there are probably only four or five white males in the United Kingdom from whom that semen stain could have come. The Defendant is one of them. If that is the position, the decision you have to reach, on all the evidence, is whether you are sure that it was the Defendant who left that stain or whether it is possible that it was one of that other small group of men who share the same DNA characteristics.
Juries should weigh up conflicting and corroborative evidence, using their own common sense and not by using mathematical formulae, such as Bayes' theorem, so as to avoid "confusion, misunderstanding and misjudgment".
In 1987, Florida rapist Tommie Lee Andrews was the first person in the United States to be convicted as a result of DNA evidence, for raping a woman during a burglary; he was convicted on 6 November 1987 and sentenced to 22 years in prison.
In 1988, Timothy Spencer was the first man in Virginia to be sentenced to death through DNA Testing for several rape and murder charges, He was dubbed "The South Side Strangler" because he killed victims on the southside of Richmond, Virginia. He was later charged with rape and 1st degree murder and was sentenced to death. He was executed on April 27, 1994. David Vasquez, initially convicted of one of Spencer's crimes, became the first man in America exonerated based on DNA evidence.
In 1991, Allan Legere was the first Canadian to be convicted as a result of DNA evidence, for four murders he had committed while an escaped prisoner in 1989. During his trial, his defense argued that the relatively shallow gene pool of the region could lead to false positives.
The science was made famous in the United States in 1994 when prosecutors heavily relied on — and through expert witnesses exhaustively presented and explained — DNA evidence allegedly linking O.J. Simpson to a double murder. The case also brought to light the laboratory difficulties and handling procedure mishaps which can cause such evidence to be significantly doubted.
In 1994, RCMP detectives successfully tested hairs from a cat known as Snowball, and used the test to link a man to the murder of his wife, thus marking for the first time in forensic history the use of non-human DNA to identify a criminal.
In 1998, Dr. Richard J. Schmidt was convicted of attempted second-degree murder when it was shown that there was a link between the viral DNA of the human immunodeficiency virus (HIV) he had been accused of injecting in his girlfriend and viral DNA from one of his patients with full-blown AIDS. This was the first time viral DNA fingerprinting had been used as evidence in a criminal trial.
In 1999, Raymond Easton a disabled man from Swindon, England was arrested and detained for 7 hours in connection with a burglary due to an inaccurrate DNA match. His DNA had been retained on file after an unrelated domestic incident some time previously.
In 2002, DNA testing was used to exonerate Douglas Echols, a man who was wrongfully convicted in a 1986 rape case. Echols was the 114th person to be exonerated through post-conviction DNA testing.
In August 2002, Annalisa Vincenzi was shot dead in Tuscany. Some time later, Bartender Peter Hamkin, 23, was arrested in Merseyside in March 2003 on an extradition warrant heard at Bow Street Magistrates' Court in London to establish whether he should be taken to Italy to face a murder charge. DNA "proved" he shot her, but he was cleared on other evidence.
In 2003, Welshman Jeffrey Gafoor was convicted of the 1988 murder of Lynette White, when crime scene evidence collected 12 years earlier was re-examined using STR techniques, resulting in a match with his nephew. This may be the first known example of the DNA of an innocent yet related individual being used to identify the actual criminal, via "familial searching".
In June 2003, because of new DNA evidence, Dennis Halstead, John Kogut and John Restivo won a re-trial on their murder conviction. The three men had already served eighteen years of their thirty-plus-year sentences.
The trial of Robert Pickton is notable in that DNA evidence is being used primarily to identify the victims, and in many cases to prove their existence.
In March 2003, Josiah Sutton was released from prison after serving four years of a twelve-year sentence for a sexual assault charge. Questionable DNA samples taken from Sutton were retested in the wake of the Houston Police Department's crime lab scandal of mishandling DNA evidence.
In 2004, DNA testing shed new light into the mysterious 1912 disappearance of Bobby Dunbar, a four-year-old boy who vanished during a fishing trip. He was allegedly found alive eight months later in the custody of William Cantwell Walters, but another woman claimed that the boy was her son, Bruce Anderson, whom she had entrusted in Walters' custody. The courts disbelieved her claim and convicted Walters for the kidnapping. The boy was raised and known as Bobby Dunbar throughout the rest of his life. However, DNA tests on Dunbar's son and nephew revealed the two were not related, thus establishing that the boy found in 1912 was not Bobby Dunbar, whose real fate remains unknown.
The 1993 rape and murder of Mia Zapata, lead singer for the Seattle punk band The Gits was unsolved 9 years after the murder. A database search in 2001 failed, but the killer's DNA was collected when he was arrested in Florida for burglary and domestic abuse in 2002.
In 2005, Gary Leiterman was convicted of the 1969 murder of Jane Mixer, a law student at the University of Michigan, after DNA found on Mixer's pantyhose was matched to Leiterman. DNA in a drop of blood on Mixer's hand was matched to John Ruelas, who was only four years old in 1969 and was never successfully connected to the case in any other way. Leiterman's defense unsuccessfully argued that the unexplained match of the blood spot to Ruelas pointed to cross-contamination and raised doubts about the reliability of the lab's identification of Leiterman.
In December 2005, Evan Simmons was proven innocent of a 1981 attack on an Atlanta woman after serving twenty-four years in prison. Mr Clark is the 164th person in the United States and the fifth in Georgia to be freed using post-conviction DNA testing.