Genetic fingerprinting
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Genetic fingerprinting, DNA testing, DNA typing, and DNA profiling are techniques used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. Two humans will have the vast majority of their DNA sequence in common. Genetic fingerprinting exploits highly variable repeating sequences called minisatellites. Two unrelated humans will be likely to have different numbers of minisatellites at a given locus. In STR profiling, which is distinct from DNA fingerprinting, PCR is used to obtain enough DNA to then detect the number of repeats at several loci. It is possible to establish a match that is extremely unlikely to have arisen by coincidence, except in the case of identical twins, who will have identical genetic profiles.
Genetic fingerprinting is used in forensic science, to match suspects to samples of blood, hair, saliva or semen. It has also led to several exonerations of formerly convicted suspects. It is also used in such applications as identifying human remains, paternity testing, matching organ donors, studying populations of wild animals, and establishing the province or composition of foods. It has also been used to generate hypotheses on the pattern of the human diaspora in prehistoric times.
Testing is subject to the legal code of the jurisdiction in which it is performed. Usually the testing is voluntary, but it can be made compulsory by such instruments as a search warrant or court order. Several jurisdictions have also begun to assemble databases containing DNA information of convicts.
The United Kingdom currently has the most extensive DNA database in the world, with well over 2 million records as of 2005: The National DNA Database (NDNAD). The size of this database,and its rate of growth, is giving concern to civil liberties groups in the UK, where police have wide-ranging powers to take samples and retain them even in the event of acquittal.
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[edit] Reference samples
DNA identification must be done by an extraction of DNA from substances such as:
- Personal items (e.g. toothbrush, razor, ...)
- Banked samples (e.g. banked sperm or biopsy tissue)
- Blood kin (biological relative)
- Human remains previously identified
Reference samples are often collected using buccal swab.
[edit] DNA fingerprinting methods
DNA fingerprinting begins by extracting DNA from the cells in a sample of blood, saliva, semen, or other appropriate fluid or tissue.
[edit] RFLP analysis
When DNA fingerprinting first began, restriction fragment length polymorphism (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-labeled 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. Many labs developed policies that described what they considered a unique band, but it was not standardized and led to DNA fingerprinting coming under harsh attack in People v. Castro 545 N.Y.S. 2d. 985 (Sup. Ct. 1989). 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.
[edit] PCR analysis
With the invention of the polymerase chain reaction (PCR), DNA fingerprinting took huge strides forward in both discriminating power and ability to recover information from very small starting samples. PCR involves the amplification of specific regions of DNA using a cycling of temperature and a thermostable polymerase enzyme along with sequence specific primers of DNA. 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.
One of the primary complaints against RFLP was that it was slow and required large quantities of DNA to be used. This led to the development of PCR-based methods which required smaller amounts of DNA that could also be more degraded than those used in RFLP analysis. Systems 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.
[edit] AmpFLP
Another technique, AmpFLP, or amplified fragment length polymorphism was also put into practice during the early 1990's. 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.
[edit] STR analysis
The most prevalent method of DNA fingerprinting 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 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 (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 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.
From country to country different STR based DNA profiling systems are in use. In North America CODIS is prevalent, while in the UK the SGM+ system, which is compatible with The National DNA Database is 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, something 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 STRs is in its statistical power of discrimination. In the U.S.A., there are 13 loci (DNA locations) that are currently used for discrimination. 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 power rule of statistics 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.
[edit] Y-chromosome analysis
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. Y-chromosomes are paternally inherited, so Y-STR analysis can help in the identification of paternally related males. Y-STR analysis was performed in the Sally Hemings controversy to determine if Thomas Jefferson had sired a son with one of his slaves.
[edit] Mitochondrial analysis
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.
[edit] Considerations when evaluating DNA evidence
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 innocence 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). However, 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 [1]. Because of this, arbitrary low ceilings were put on match probabilities used in RFLP analysis rather than the higher theoretically computed ones [2]. Today, RFLP has become widely disused due to these difficulties in interpretation.
STRs do not suffer from such subjectivity and provide much better powers of discrimination, for unrelated individuals (of the order of 1 in 10^29 if using a full profile) It should be noted that figures of this magnitude are not considered to be statistically suportable 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, this test updated the SGM DNA profiling system on which the National DNA Database was founded in 1995. The SGM system included 6 out of the 10 STR regions used in the SGM+ system and the same sex indicating test, however the discriminating power of the SGM system was only considered to be supportable at 1 in a million) . 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.
When evaluating a DNA match, the following questions should be asked:
- Could it be an accidental random match?
- If not, could the DNA sample have been planted?
- If not, did the accused leave the DNA sample at the exact time of the crime?
- If yes, does that mean that the accused is guilty of the crime?
[edit] Fake DNA evidence
The value of DNA evidence has to be seen in light of recent cases where criminals planted fake DNA samples at crime scenes. In one case, a criminal even planted fake DNA evidence in his own body: Dr. John Schneeberger of Canada raped one of his sedated patients in 1992 and left semen on her underwear. Police drew Schneeberger's blood and compared its DNA against the crime scene semen DNA on three occasions, never showing a match. It turned out that he had surgically inserted a Penrose drain into his arm and filled it with foreign blood and anticoagulants.
[edit] Cases
In the 1920s, Anna Anderson claimed that she was Princess Anastasia Romanov of Russia; in the 1980s her cremated remains were tested and seemed to show that she was no relation to the Romanovs.
In 1987, British baker Colin Pitchfork was the first criminal caught using DNA fingerprinting in Leicester, the city where it was first discovered.
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. [3] [4]
In 1989, Chicago man Gary Dotson was the first person whose conviction was overturned using 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.
In 1992, DNA evidence was used to prove that Nazi doctor Josef Mengele was buried in Brazil under the name Wolfgang Gerhard.
In 1993, Kirk Bloodsworth was the first person to have been convicted of murder and sentenced to death, whose conviction was overturned using DNA evidence.
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 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 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.[5] 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 of 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 December 2005, Robert Clark 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 United States and the fifth in Georgia to be freed using post-conviction DNA testing.
[edit] See also
- National DNA Database
- restriction fragment length polymorphism (RFLP)
- paternity test
- genealogical DNA test
- short tandem repeat (STR)
- capillary electrophoresis (CE)
- Gene mapping
- Harvey v. Horan
