DNA profiling

DNA profiling (also called DNA fingerprinting) is the process of determining an individual's DNA characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding. DNA profiling is a forensic technique in criminal investigations, comparing criminal suspects' profiles to DNA evidence so as to assess the likelihood of their involvement in the crime. It is also used in parentage testing, to establish immigration eligibility, and in genealogical and medical research. DNA profiling has also been used in the study of animal and plant populations in the fields of zoology, botany, and agriculture. Background: Starting in the 1980s scientific advances allowed the use of DNA as a material for the identification of an individual. The first patent covering the direct use of DNA variation for forensics was filed by Dr. Jeffrey Glassberg in 1983, based upon work he had done while at Rockefeller University in 1981. In the United Kingdom, Geneticist Sir Alec Jeffreys independently developed a DNA profiling process in beginning in late 1984 while working in the Department of Genetics at the University of Leicester. The process, developed by Jeffreys in conjunction with Peter Gill and Dave Werrett of the Forensic Science Service (FSS), was first used forensically in the solving of the murder of two teenagers who had been raped and murdered in Narborough, Leicestershire in 1983 and 1986. In the murder inquiry, led by Detective David Baker, the DNA contained within blood samples obtained voluntarily from around 5,000 local men who willingly assisted Leicestershire Constabulary with the investigation, resulted in the exoneration of a man who had confessed to one of the crimes, and the subsequent conviction of Colin Pitchfork. Pitchfork, a local bakery employee, had coerced his coworker Ian Kelly to stand in for him when providing a blood sample—Kelly then used a forged passport to impersonate Pitchfork. Another coworker reported the deception to the police. Pitchfork was arrested, and his blood was sent to Jeffrey's lab for processing and profile development. Pitchfork's profile matched that of DNA left by the murderer which confirmed Pitchfork's presence at both crime scenes; he pleaded guilty to both murders. Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different that it is possible to distinguish one individual from another, unless they are monozygotic (identical) twins. DNA profiling uses repetitive sequences that are highly variable, called variable number tandem repeats (VNTRs), in particular short tandem repeats (STRs), also known as microsatellites, and minisatellites. VNTR loci are similar between closely related individuals, but are so variable that unrelated individuals are unlikely to have the same VNTRs. In India DNA fingerprinting was started by Dr. VK Kashyap and Dr. Lalji Singh. Dr.Singh was an Indian scientist who worked in the field of DNA fingerprinting technology in India, where he was popularly known as the "Father of Indian DNA fingerprinting". Profiling processes: The process, developed by Glassberg and independently by Jeffreys, begins with a sample of an individual's DNA (typically called a "reference sample"). Reference samples are usually collected through a buccal swab. When this is unavailable (for example, when a court order is needed but unobtainable) other methods may be needed to collect a sample of blood, saliva, semen, vaginal lubrication, or other fluid or tissue from personal use items (for example, a toothbrush, razor) or from stored samples (for example, banked sperm or biopsy tissue). Samples obtained from blood relatives can indicate an individual's profile, as could previous profiled human remains. A reference sample is then analyzed to create the individual's DNA profile using one of the techniques discussed below. The DNA profile is then compared against another sample to determine whether there is a genetic match. DNA extraction: When a sample such as blood or saliva is obtained, the DNA is only a small part of what is present in the sample. Before the DNA can be analyzed, it must be extracted from the cells and purified. There are many ways this can be accomplished, but all methods follow the same basic procedure. The cell and nuclear membranes need to be broken up to allow the DNA to be free in solution. Once the DNA is free, it can be separated from all other cellular components. After the DNA has been separated in solution, the remaining cellular debris can then be removed from the solution and discarded, leaving only DNA. The most common methods of DNA extraction include organic extraction (also called phenol chloroform extraction), Chelex extraction, and solid phase extraction. Differential extraction is a modified version of extraction in which DNA from two different types of cells can be separated from each other before being purified from the solution. Each method of extraction works well in the laboratory, but analysts typically selects their preferred method based on factors such as the cost, the time involved, the quantity of DNA yielded, and the quality of DNA yielded. After the DNA is extracted from the sample, it can be analyzed, whether it is by RFLP analysis or quantification and PCR analysis. RFLP analysis: The first methods for finding out genetics used for DNA profiling involved RFLP analysis. DNA is collected from cells and cut into small pieces using a restriction enzyme (a restriction digest). This generates DNA fragments of differing sizes as a consequence of variations between DNA sequences of different individuals. The fragments are then separated on the basis of size using gel electrophoresis. The separated fragments are then transferred on to a nitrocellulose or nylon filter; this procedure is called a Southern blot. The DNA fragments within the blot are permanently fixed to the filter, and the DNA strands are denatured. Radiolabeled probe molecules are then added that are complementary to sequences in the genome that contain repeat sequences. These repeat sequences tend to vary in length among different individuals and are called variable number tandem repeat sequences or VNTRs. The probe molecules hybridize to DNA fragments containing the repeat sequences and excess probe molecules are washed away. The blot is then exposed to an X-ray film. Fragments of DNA that have bound to the probe molecules appear as fluorescent bands on the film. The Southern blot technique requires large amounts of non-degraded sample DNA. Also, Alec Jeffrey's original multilocus RFLP 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 paternity testing). These early techniques have been supplanted by PCR-based assays. Polymerase chain reaction (PCR) analysis: Developed by Kary Mullis in 1983, a process was reported by which specific portions of the sample DNA can be amplified almost indefinitely (Saiki et al. 1985, 1985) The process, polymerase chain reaction (PCR), mimics the biological process of DNA replication, but confines it to specific DNA sequences of interest. With the invention of the 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. In the PCR process, the DNA sample is denatured into the separate individual polynucleotide strands through heating. Two oligonucleotide DNA primers are used to hybridize to two corresponding nearby sites on opposite DNA strands in such a fashion that the normal enzymatic extension of the active terminal of each primer (that is, the 3’ end) leads toward the other primer. PCR uses replication enzymes that are tolerant of high temperatures, such as the thermostable Taq polymerase. In this fashion, two new copies of the sequence of interest are generated. Repeated denaturation, hybridization, and extension in this fashion produce an exponentially growing number of copies of the DNA of interest. Instruments that perform thermal cycling are readily available from commercial sources. This process can produce a million-fold or greater amplification of the desired region in 2 hours or less. Early assays such as the HLA-DQ alpha reverse dot blot strips grew to be very popular owing to their ease of use, and the speed with which a result could be obtained. However, they were not as discriminating as RFLP analysis. It was also difficult to determine a DNA profile for mixed samples, such as a vaginal swab from a sexual assault victim. However, the PCR method was readily adaptable for analyzing VNTR, in particular STR loci. In recent years, research in human DNA quantitation has focused on new "real-time" quantitative PCR (qPCR) techniques. Quantitative PCR methods enable automated, precise, and high-throughput measurements. Inter-laboratory studies have demonstrated the importance of human DNA quantitation on achieving reliable interpretation of STR typing and obtaining consistent results across laboratories. STR analysis: The system of DNA profiling used today is based on polymerase chain reaction (PCR) and uses simple sequences or 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 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 derives from inspecting 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 that amplify the CODIS 20 core loci are almost universal, whereas in the United Kingdom the DNA-17 17 loci system (which is compatible with The National DNA Database) is in use, and Australia uses 18 core markers. 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 20 loci that are currently used for discrimination in CODIS are independently assorted (having a certain number of repeats at one locus does not change the likelihood of having any number of repeats at any other locus), the product rule for probabilities can be applied. This means that, if someone has the DNA type of ABC, where the three loci were independent, then the probability of that individual having that DNA type is the probability of having type A times the probability of having type B times the probability of having type C. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1x1018) or more. However, DNA database searches showed much more frequent than expected false DNA profile matches. 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 contamination of a sample from nearby objects, or from left-over cells transferred from a prior test. 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 in order 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 was not actually matched as being related to their own DNA in another sample. AFLP: Another technique, AFLP, 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 focus 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. DNA family relationship analysis: 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. There are predictable inheritance patterns at certain locations (called loci) in the human genome, which have been found to be useful in determining identity and biological relationships. These loci contain specific DNA markers that 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 their 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. 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 enable a conclusion to be drawn as to whether or not the man is the biological father. 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 a randomly selected 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. Y-chromosome analysis: 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 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 Jefferson-Hemings controversy to determine if Thomas Jefferson had sired a son with one of his slaves. The analysis of the Y-chromosome yields weaker results than autosomal chromosome analysis with regard to individual identification. The Y male sex-determining chromosome, as it is inherited only by males from their fathers, is almost identical along the paternal line. On the other hand, the Y-STR haplotype provides powerful genealogical information as a patrilinear relationship can be traced back over many generations. Furthermore, due to the paternal inheritance, Y-haplotypes provide information about the genetic ancestry of the male population. To investigate this population history, and to provide estimates for haplotype frequencies in criminal casework, the "Y haplotype reference database (YHRD)" has been created in 2000 as an online resource. It currently comprises more than 300,000 minimal (8 locus) haplotypes from world-wide populations. 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, and 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. In general, a difference of two or more nucleotides is 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. Control mechanism based on interaction point with data. This can be determined by tooled placement in sample. Issues with forensic DNA samples: When people think of DNA analysis they often think about shows like NCIS or CSI, which portray DNA samples coming into a lab and then instantly analyzed, followed by pulling up a picture of the suspect within minutes⁠. The true reality, however, is quite different and perfect DNA samples are often not collected from the scene of a crime. Homicide victims are frequently left exposed to harsh conditions before they are found and objects used to commit crimes have often been handled by more than one person. The two most prevalent issues that forensic scientists encounter when analyzing DNA samples are degraded samples and DNA mixtures. Degraded DNA: In the real world DNA labs often have to deal with DNA samples that are less than ideal. DNA samples taken from crime scenes are often degraded, which means that the DNA has started to break down into smaller fragments. Victims of homicides might not be discovered right away, and in the case of a mass casualty event it could be hard to get DNA samples before the DNA has been exposed to degradation elements. Degradation or fragmentation of DNA at crime scenes can occur because of a number of reasons, with environmental exposure often being the most common cause. Biological samples that have been exposed to the environment can get degraded by water and enzymes called nucleases. Nucleases essentially ‘chew’ up the DNA into fragments over time and are found everywhere in nature. Before modern PCR methods existed it was almost impossible to analyze degraded DNA samples. Methods like restriction fragment length polymorphism or RFLP Restriction fragment length polymorphism, which was the first technique used for DNA analysis in forensic science, required high molecular weight DNA in the sample in order to get reliable data. High molecular weight DNA however is something that is lacking in degraded samples, as the DNA is too fragmented to accurately carry out RFLP. It wasn't until modern day PCR techniques were invented that analysis of degraded DNA samples were able to be carried out Polymerase chain reaction. Multiplex PCR in particular made it possible to isolate and amplify the small fragments of DNA still left in degraded samples. When multiplex PCR methods are compared to the older methods like RFLP a vast difference can be seen. Multiplex PCR can theoretically amplify less than 1 ng of DNA, while RFLP had to have a least 100 ng of DNA in order to carry out an analysis. In terms of a forensic approach to a degraded DNA sample, STR loci STR analysis are often amplified using PCR-based methods. Though STR loci are amplified with greater probability of success with degraded DNA, there is still the possibility that larger STR loci will fail to amplify, and therefore, would likely yield a partial profile, which results in reduced statistical weight of association in the event of a match. MiniSTR Analysis: In instances where DNA samples are degraded, like in the case of intense fires or if all that remains are bone fragments, standard STR testing on these samples can be inadequate. When standard STR testing is done on highly degraded samples the larger STR loci often drop out, and only partial DNA profiles are obtained. While partial DNA profiles can be a powerful tool, the random match probabilities will be larger than if a full profile was obtained. One method that has been developed in order to analyse degraded DNA samples is to use miniSTR technology. In this new approach, primers are specially designed to bind closer to the STR region. In normal STR testing the primers will bind to longer sequences that contain the STR region within the segment. MiniSTR analysis however will just target the STR location, and this results in a DNA product that is much smaller. By placing the primers closer to the actual STR regions, there is a higher chance that successful amplification of this region will occur. Successful amplification of these STR regions can now occur and more complete DNA profiles can be obtained. The success that smaller PCR products produce a higher success rate with highly degraded samples was first reported in 1995, when miniSTR technology was used to identify victims of the Waco fire. In this case the fire at destroyed the DNA samples so badly that normal STR testing did not result in a positive ID on some of the victims. DNA Mixtures: Mixtures are another common issue that forensic scientists face when they are analyzing unknown or questionable DNA samples. A mixture is defined as a DNA sample that contains two or more individual contributors. This can often occur when a DNA sample is swabbed from an item that is handled by more than one person or when a sample contains both the victim and assailants' DNA. The presence of more than one individual in a DNA sample can make it challenging to detect individual profiles, and interpretation of mixtures should only be done by highly trained individuals. Mixtures that contain two or three individuals can be interpreted, though it will be difficult. Mixtures that contain four or more individuals are much too convoluted to get individual profiles. One common scenario in which a mixture is often obtained is in the case of sexual assault. A sample may be collected that contains material from the victim, the victim's consensual sexual partners, and the perpetrator(s). As detection methods in DNA profiling advance, forensic scientists are seeing more DNA samples that contain mixtures, as even the smallest contributor is now able to be detected by modern tests. The ease in which forensic scientists have in interpenetrating DNA mixtures largely depends on the ratio of DNA present from each individual, the genotype combinations, and total amount of DNA amplified. The DNA ratio is often the most important aspect to look at in determining whether a mixture can be interpreted. For example, in the case where a DNA sample had two contributors, it would be easy to interpret individual profiles if the ratio of DNA contributed by one person was much higher than the second person. When a sample has three or more contributors, it becomes extremely difficult to determine individual profiles. Fortunately, advancements in probabilistic genotyping could make this sort of determination possible in the future. Probabilistic genotyping uses complex computer software to run through thousands of mathematical computations in order to produce statistical likelihoods of individual genotypes found in a mixture. Probabilistic genotyping software that are often used in labs today include STRmix and TrueAllele.