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 by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier. DNA profiling should not be confused with full genome sequencing.[1] It is used in, for example, parental testing and criminal investigation.
Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different to distinguish one individual from another, unless they are monozygotic twins.[2] DNA profiling uses repetitive ("repeat") sequences that are highly variable,[2] called variable number tandem repeats (VNTRs), particularly short tandem repeats (STRs). VNTR loci are very similar between closely related humans, but so variable that unrelated individuals are extremely unlikely to have the same VNTRs.
The DNA profiling technique was first reported in 1984[3] by Sir Alec Jeffreys at the University of Leicester in England,[4] and is now the basis of several national DNA databases. Dr. Jeffreys's genetic fingerprinting was made commercially available in 1987, when a chemical company, Imperial Chemical Industries (ICI), started a blood-testing centre in England.[5]
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The process begins with a sample 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 (e.g. 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 analyzed to create the individual's DNA profile using one of a number of techniques, discussed below. The DNA profile is then compared against another sample to determine whether there is a genetic match.
The first methods for finding out genetics 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, Karl Brown's 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 was readily adaptable for analyzing VNTR, particularly STR loci.
The method of DNA profiling used today is based on PCR and uses short tandem repeats (STR) a type of VNTR. 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 unrelated people almost certainly have different numbers of repeat units, STRs can be used to discriminate between unrelated individuals. These STR loci (locations on a chromosome) are targeted with sequence-specific primers and 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.
Each STR is polymorphic, but the number of alleles is very small. Typically each STR allele will be shared by around 5 - 20% of individuals. The power of STR analysis comes from looking at multiple STR loci simultaneously. The pattern of alleles can identify an individual quite accurately. Thus STR analysis provides an excellent 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+ 10 loci system (which is compatible with The National DNA Database), is in use. Whichever system is used, many of the STR regions used are the same. These DNA-profiling systems are based on multiplex reactions, whereby many STR regions will be tested at the same time.
The true power of STR analysis is in its statistical power of discrimination. Because the 13 loci that are currently used for discrimination in CODIS 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 (1x1018) or more. However, DNA database searches showed much more frequent than expected false DNA profile matches.[6] Moreover, since there are about 12 million monozygotic twins on Earth, the theoretical probability is not accurate.
In practice, the risk of contaminated-matching is much greater than matching a distant relative, such as a sample being contaminated from nearby objects, or from left-over cells transferred from a prior test. Logically, the risk is greater for matching the most common person in the samples: everything collected from, or in contact with, a victim is a major source of contamination for any other samples brought into a lab. For that reason, multiple control-samples are typically tested, to ensure that they stayed clean, when prepared during the same period as the actual test samples. Unexpected matches (or variations) in several control-samples indicates a high probability of contamination for the actual test samples. In a relationship test, the full DNA profiles should differ (except for twins), to prove that a person wasn't actually matched as being related to their own DNA in another sample.
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.
Using PCR technology, DNA analysis is widely applied to determine genetic family relationships such as paternity, maternity, siblingship and other kinships.
During conception, the father’s sperm cell and the mother’s egg cell, each containing half the amount of DNA found in other body cells, meet and fuse to form a fertilized egg, called a zygote. The zygote contains a complete set of DNA molecules, a unique combination of DNA from both parents. This zygote divides and multiplies into an embryo and later, a full human being.
At each stage of development, all the cells forming the body contain the same DNA—half from the father and half from the mother. This fact allows the relationship testing to use all types of all samples including loose cells from the cheeks collected using buccal swabs, blood or other types of samples.
While a lot of DNA contains information for a certain function, there is some called junk DNA, which is currently used for human identification. At some special locations (called loci) in the junk DNA, predictable inheritance patterns were found to be useful in determining biological relationships. These locations contain specific DNA markers that DNA scientists use to identify individuals. In a routine DNA paternity test, the markers used are Short Tandem Repeats (STRs), short pieces of DNA that occur in highly differential repeat patterns among individuals.
Each person’s DNA contains two copies of these markers—one copy inherited from the father and one from the mother. Within a population, the markers at each person’s DNA location could differ in length and sometimes sequence, depending on the markers inherited from the parents.
The combination of marker sizes found in each person makes up his/her unique genetic profile. When determining the relationship between two individuals, their genetic profiles are compared to see if they share the same inheritance patterns at a statistically conclusive rate.
For example, the following sample report from this commercial DNA paternity testing laboratory Universal Genetics signifies how relatedness between parents and child is identified on those special markers:
| DNA Marker | Mother | Child | Alleged father |
|---|---|---|---|
| D21S11 | 28, 30 | 28, 31 | 29, 31 |
| D7S820 | 9, 10 | 10, 11 | 11, 12 |
| TH01 | 14, 15 | 14, 16 | 15, 16 |
| D13S317 | 7, 8 | 7, 9 | 8, 9 |
| D19S433 | 14, 16.2 | 14, 15 | 15, 17 |
The partial results indicate that the child and the alleged father’s DNA match among these five markers. The complete test results show this correlation on 16 markers between the child and the tested man to draw a conclusion of whether or not the man is the biological father.
Scientifically, each marker is assigned with a Paternity Index (PI), which is a statistical measure of how powerfully a match at a particular marker indicates paternity. The PI of each marker is multiplied with each other to generate the Combined Paternity Index (CPI), which indicates the overall probability of an individual being the biological father of the tested child relative to any random man from the entire population of the same race. The CPI is then converted into a Probability of Paternity showing the degree of relatedness between the alleged father and child.
The DNA test report in other family relationship tests, such as grandparentage and siblingship tests, is similar to a paternity test report. Instead of the Combined Paternity Index, a different value, such as a Siblingship Index, is reported.
The report shows the genetic profiles of each tested person. If there are markers shared among the tested individuals, the probability of biological relationship is calculated to determine how likely the tested individuals share the same markers due to a blood relationship.
Recent innovations have included the creation of primers targeting polymorphic regions on the Y-chromosome (Y-STR), which allows resolution of a mixed DNA sample from a male and female and/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.
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 daughter'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 clear identities, such as those of missing people 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.
There are now several DNA databases in existence around the world. Some are private, but most of the largest databases are government controlled. The United States maintains the largest DNA database, with the Combined DNA Index System, holding over 5 million records as of 2007.[7] The United Kingdom maintains the National DNA Database (NDNAD), which is of similar size, despite the UK's smaller population. 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.[8]
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.[9]
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), 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.[10] 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.[11] Today, RFLP has become widely disused due to the advent of more discriminating, sensitive and easier technologies.
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. STRs do not suffer from such subjectivity and provide similar power of discrimination (1 in 1013 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 is considered statistically supportable. 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.
It's also possible to use DNA profiling as evidence of genetic relationship, but testing that shows no relationship is absolutely certain. While almost all individuals have a single and distinct set of genes, rare individuals, known as "chimeras", have at least two different sets of genes. There have been several cases of DNA profiling that falsely suggested that a mother was unrelated to her children.[12]
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 raped one of his sedated patients in 1992 and left semen on her underwear. Police drew what they believed to be 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.
The functional analysis of genes and their coding sequences (open reading frames [ORFs]) typically requires that each ORF be expressed, the encoded protein purified, antibodies produced, phenotypes examined, intracellular localization determined, and interactions with other proteins sought.[13] In a study conducted by the life science company Nucleix and published in the journal Forensic Science International, scientists found that an In vitro synthesized sample of DNA matching any desired genetic profile can be constructed using standard molecular biology techniques without obtaining any actual tissue from that person.
In the case of the Phantom of Heilbronn, police detectives found DNA traces from the same woman on various crime scenes in Austria, Germany and France - among them murders, burglaries and robberies. Only after the DNA of the "woman" matched the DNA sampled from the burned body of a male asylum seeker in France, detectives began to have serious doubts about the DNA evidence. In that case, DNA traces were already present on the cotton swabs used to collect the samples at the crime scene, and the swabs had all been produced at the same factory in Austria. The company's product specification said that the swabs were guaranteed to be sterile, but not DNA-free.
Familial DNA searching (sometimes referred to as “Familial DNA” or “Familial DNA Database Searching”) is the practice of creating new investigative leads in cases where DNA evidence found at the scene of a crime (forensic profile) strongly resembles that of an existing DNA profile (offender profile) in a state DNA database but there is not an exact match.[14][15] After all other leads have been exhausted, investigators may use specially developed software to compare the forensic profile to all profiles taken from a state’s DNA database in order to generate a list of those offenders most likely to be a close relative of the forensic profile.[16] To eliminate the majority of this list, crime lab technicians conduct Y-STR analysis that confirms the familial relationships suggested by the first list. Using standard investigative techniques, authorities are then able to build a family tree. The family tree is populated from information gathered from public records and criminal justice records. Investigators rule out family members’ involvement in the crime by finding excluding factors such as sex, living out of state or being incarcerated when the crime was committed. They may also use other leads from the case, such as witness or victim statements, to identify a suspect. Once a suspect has been identified, investigators seek to legally obtain a DNA sample from the suspect. This suspect DNA profile is then compared to the sample found at the crime scene, in accordance with well established and constitutionally accepted practices, to definitively identify the suspect as the source of the crime scene DNA.
Familial DNA database searching was first used to convict Craig Harman of manslaughter in the United Kingdom on April 19, 2004.[17] Currently, familial DNA database searching is not conducted on a national level in the United States. States determine their own policies and decision making processes for how and when to conduct familial searches. The first familial DNA search and subsequent conviction in the United States was conducted in Denver, Colorado in 2008 using software developed under the leadership of Denver District Attorney Mitch Morrissey and Denver Police Department Crime Lab Director Gregg LaBerge.[18] California was the first state to implement a policy for familial searching under then Attorney General, now Governor, Jerry Brown.[19] In his role as consultant to the Familial Search Working Group of the California Department of Justice, former Alameda County Prosecutor Rock Harmon is widely considered to have been the catalyst in the adoption of familial search technology in California. The technique was used to catch the Los Angeles serial killer known as the “Grim Sleeper” in 2010[20] and more recently led to the arrest of 21-year-old Elvis Garcia on charges of sexual assault and false imprisonment of a woman in Santa Cruz in 2008.[21] In March 2011 Virginia Governor Bob McDonnell announced that Virginia would begin using familial DNA searches.[22] Other states are expected to follow.
At a press conference in Virginia on March 7, 2011 regarding the East Coast Rapist, Prince William County prosecutor Paul Ebert and Fairfax County Police Detective John Kelly said the case would have been solved years ago if Virginia had used familial DNA searching. Aaron Thomas, the suspected East Coast Rapist, was arrested in connection with the rape of 17 women from Virginia to Rhode Island, but familial DNA was not used in the case.[23]
Critics of familial DNA database searches argue that the technique may be an invasion of an individual’s 4th Amendment rights.[24] Some scholars have pointed out that the privacy concerns surrounding familial searching are no more threatening than other police search techniques.[25] The Ninth Circuit Court of Appeals in United States v. Pool ruled that this practice is somewhat analogous to a witness looking at a photograph of one person and stating that it looked like the perpetrator, which leads law enforcement to show the witness photos of similar looking individuals, one of whom is identified as the perpetrator.[26] Regardless of whether familial DNA searching was the method used to identify the suspect, authorities always conduct a normal DNA test to match the suspect’s DNA with that of the DNA left at the crime scene.
For instance, investigators with Denver District Attorney’s Office successfully identified a suspect in a property theft case using a familial DNA search. In this example, the suspect’s blood left at the scene of the crime strongly resembled that of a current Colorado Department of Corrections prisoner.[27] Using publicly available records, the investigators created a family tree. They then eliminated all the family members who were incarcerated at the time of the offense, as well as all of the females (the crime scene DNA profile was that of a male). Investigators obtained a court order to collect the suspect’s DNA, but the suspect actually volunteered to come to a police station and give a DNA sample. After providing the sample, the suspect walked free without further interrogation or detainment. Later confronted with an exact match to the forensic profile, the suspect pled guilty to criminal trespass at the first court date and was sentenced to two years probation.
Partial DNA matches are not searches themselves, but are the result of moderate stringency CODIS searches that produce a potential match that shares at least one allele at every locus.[28] Partial matching does not involve the use of a familial search software, such as those used in the UK and Denver, or additional Y-STR analysis, and therefore often misses sibling relationships. Partial matching has been used to identify suspects in several cases in the UK and US[29] and has also been used as a tool to exonerate the falsely accused. Darryl Hunt was wrongly convicted in connection with the rape and murder of a young woman in 1984 in North Carolina.[30] Hunt was exonerated in 2004 when a DNA database search produced a remarkably close match between a convicted felon and the forensic profile from the case. The partial match led investigators to the felon’s brother, Willard E. Brown, who confessed to the crime when confronted by police. A judge then signed an order to dismiss the case with prejudice against Hunt, ending the long ordeal for this innocent man.
Police forces may collect DNA samples without the suspects' knowledge, and use it as evidence. Legality of this mode of proceeding has been questioned in Australia.
In the United States, it has been accepted, courts often claiming that there was no expectation of privacy, citing California v. Greenwood (1985), during which the Supreme Court held that the Fourth Amendment does not prohibit the warrantless search and seizure of garbage left for collection outside the curtilage of a home. Critics of this practice underline the fact that this analogy ignores that "most people have no idea that they risk surrendering their genetic identity to the police by, for instance, failing to destroy a used coffee cup. Moreover, even if they do realize it, there is no way to avoid abandoning one’s DNA in public."[31]
In the UK, the Human Tissue Act 2004 prohibited private individuals from covertly collecting biological samples (hair, fingernails, etc.) for DNA analysis, but excluded medical and criminal investigations from the offence.[32]
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.[33] The judge must ensure that the jury must understand the significance of DNA matches and mismatches in the profiles. The judge must also ensure that the jury does not confuse the 'match probability' (the probability that a person that is chosen 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 1996 R v. Doheny[34] 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".[35]
In R v Bates,[36] Moore-Bick LJ said:
There are state laws on DNA profiling in all 50 states of the United States.[38] Detailed information on database laws in each state can be found at the National Conference of State Legislatures website.[39]
In August 2009, scientists in Israel raised serious doubts concerning the use of DNA by law enforcement as the ultimate method of identification. In a paper published in the journal Forensic Science International: Genetics, the Israeli researchers demonstrated that it is possible to manufacture DNA in a laboratory, thus falsifying DNA evidence. The scientists fabricated saliva and blood samples, which originally contained DNA from a person other than the supposed donor of the blood and saliva.[40]
The researchers also showed that, using a DNA database, it is possible to take information from a profile and manufacture DNA to match it, and that this can be done without access to any actual DNA from the person whose DNA they are duplicating. The synthetic DNA oligos required for the procedure are common in molecular laboratories.[40]
The New York Times quoted the lead author on the paper, Dr. Daniel Frumkin, saying, "You can just engineer a crime scene... any biology undergraduate could perform this."[40]
Dr. Frumkin perfected a test that can differentiate real DNA samples from fake ones. His test detects epigenetic modifications, in particular, DNA methylation. Seventy percent of the DNA in any human genome is methylated, meaning it contains methyl group modifications within a CpG dinucleotide context. Methylation at the promoter region is associated with gene silencing. The synthetic DNA lacks this epigenetic modification, which allows the test to distinguish manufactured DNA from original, genuine, DNA.[40]
It is unknown how many police departments, if any, currently use the test. No police lab has publicly announced that it is using the new test to verify DNA results.[41]