DNA testing

DNA testing (also called Genetic fingerprinting, DNA typing, or DNA profiling) is a technique used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Dr. Alec Jeffreys at the University of Leicester was announced in 1984. 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 unlikely to have the same 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. But not fingerprints.

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 States maintains the largest DNA database in the world: The Combined DNA Index System, with over 4.5 million records as of 2007. The United Kingdom, maintains the National DNA Database (NDNAD), which is of similar size. 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.

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.

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.

RFLP analysis
One way to fingerprint DNA is by doing a Southern blot. This has several steps. First, the DNA being analyzed must be separated from other material. Next, it must be cut into a few different-sized pieces using restriction enzymes, proteins that can cut double-stranded DNA without damaging the bases. The pieces are sorted by size through gel electrophoresis. The pieces are poured into gel with a positive charge at the bottom. DNA has a natural slightly negative charge so it will be attracted to the bottom. The smaller pieces can move more quickly through the gel, therefore they will be further toward the bottom than the larger pieces. This will separate the pieces by size, with the larger ones higher up and the smaller ones further down. Next, alkaline solution or heat is applied to the gel so that the DNA denatures and separates into single strands. Nitrocellulose paper is pressed evenly against the gel and then baked so the DNA is permanently attached to it. The DNA is now ready to be analyzed using a radioactive probe in a hybridization reaction.

To make a radioactive probe, DNA polymerase is needed. The DNA that is going to be made radioactive should be put in a tube. Horizontal breaks should be made along the strand, while at the same time nucleotides should be added. The base C, or cytosine, should be radioactive. Next, the polymerase should be added to the tube. It will be attracted to the breaks and try to fix them. As the DNA polymerase fixes the DNA, it will break the existing bonds so that the existing nucleotides can be replaced by the new nucleotides in the tube. Whenever the lower strand has a G base, or guanine, the C put in will be radioactive. By repairing the strand of DNA, the polymerase is also making it radioactive. The DNA is heated so that the two strands split. Single-stranded pieces that might or might not be radioactive are made. The radioactive pieces are now probes ready for use. Now the radioactive probe can be used to create a hybridization reaction. Hybridization is when two genetic sequences bind together because of the hydrogen bonds that are in between the base pairs. There are two of these bonds between A, or adenine, and T, or thymine, and three between C and G. To make hybridization works, the DNA has to be denatured so it is single-stranded; like the Southern Blot that was made on the nitrocellulose paper. The denatured DNA and the radioactive probe should be put into a plastic bag with saline liquid, and then shaken. The probe will bond to the denatured DNA wherever it finds a fit. The probe and the DNA do not have to fit together precisely. The two will have sequences that can stick together even if the fit is poor, however there will be fewer hydrogen bonds. Probes that have low homology, or similarity, can bind to the DNA better if the temperature is varied or the amount of salt in the mixture is changed. Even if the fit is poor, the probe and the DNA are now hybridized. A way to make use of the whole process described above is by using it to determine a person’s VNTRs. VNTRs, or Variable Number Tandem Repeats, are repeated sequences of base pairs in someone’s genetic information. Every DNA strand contains exons, or sections that have genetic information, and introns, which have no discernible use other than containing VNTRs, or repeating sequences of base pairs. Every single human being has a few of these repeating sequences. To find out if somebody has a specific VNTR, a Southern Blot must be made, and then probed in a hybridization reaction, by a radioactive version of said VNTR. This process ends up making a pattern called a DNA fingerprint. Every person has VNTRs they have inherited genetically from one or both parents. It is impossible for somebody to have one that neither of their parents did. VNTR patterns are unique for each person, and they will be more exact if more VNTR probes are used.'''

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 flourescently labelled 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.

AmpFLP
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.

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 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, 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 STR analysis is in its statistical power of discrimination. In the U.S.A., 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.

At least, that is the theory. The problem is ... we humans do not mate randomly. Within the relevant small subpopulation, the probabilities of the different types could be very very different from what they are in the whole population. Instead of multiplying 13 times a smallish probability together, and coming up with one in a quintillion, probably one should multiply together 13 times a quite large but quite unknown probability - resulting is something like 1 in 10, or 1 in a thousand? No-one knows. There is presently much concern among scientists that the lack of understanding of DNA evidence among police, lawyers, and judges, has already caused serious miscarriages of justice in many countries. DNA evidence is useful for showing that someone could not be guilty (when you have a mismatch), but its value in proving that someone is guilty, is basically dubious, and unknown.

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.

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.

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 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). 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. 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.

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?

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.

England
Evidence from an expert who has compared DNA samples must be accompanied by evidence as to the sources of the samples and the procedures for obtaining the DNA profiles. The judge must ensure that the jury understand the significance of matches and mismatches in the profiles. The judge must also ensure that the jury do not confuse the 'match probability' (the probability that a person picked at random has a matching DNA profile to the sample from the scene) with the 'likelihood ratio' (the probability that a person with matching DNA committed the crime). In Phillips LJ gave this example of a summing up, which should be carefully tailored to the particular facts in each case: "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".

Presentation and evaluation of evidence of partial or incomplete DNA profiles
R v Bates (2006) EWCA Crim 1395 Moore-Bick LJ said:
 * “We can see no reason why partial profile DNA evidence should not be admissible provided that the jury are made aware of its inherent limitations and are given a sufficient explanation to enable them to evaluate it. There may be cases where the match probability in relation to all the samples tested is so great that the judge would consider its probative value to be minimal and decide to exclude the evidence in the exercise of his discretion, but this gives rise to no new question of principle and can be left for decision on a case by case basis. However, the fact that there exists in the case of all partial profile evidence the possibility that a "missing" allele might exculpate the accused altogether does not provide sufficient grounds for rejecting such evidence. In many cases there is a possibility (at least in theory) that evidence exists which would assist the accused and perhaps even exculpate him altogether, but that does not provide grounds for excluding relevant evidence that is available and otherwise admissible, though it does make it important to ensure that the jury are given sufficient information to enable them to evaluate that evidence properly”.

Cases
In the 1920s, Anna Anderson claimed that she was Grand Duchess Anastasia Romanov of Russia; in the 1980s after her death, samples of her tissue that had been stored at a Charlottesville, Virginia hospital following a medical procedure were tested using DNA fingerprinting and showed that she bore 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. 

In 1988, Timothy Spencer was the first man in the United States to be sentenced to death through DNA Testing for several rape and murder charges, He was dubbed "The South Side Strangler" Because he killed all his 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.

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 &mdash; and through expert witnesses exhaustively presented and explained &mdash; 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 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, 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 United States and the fifth in Georgia to be freed using post-conviction DNA testing.