Genetic technologies, which burst into human life at the end of the last century, have changed our world to such an extent that it is already unimaginable without them. These technologies have also managed to penetrate into forensics; for decades genetic identification has been used as a fast and relatively cheap method that allows finding criminals and solving their acts without leaving the laboratory. As a pharmacologist by training, I am very interested in genetics and am eager to study this field in its various aspects. In this publication I will introduce you to classical genetic approaches in the field of forensics.
A bit of history or what does it have to do with knights?
150 years ago Johannes Friedrich Miescher discovered nucleic acids, a concept that eventually turned the world upside down [1]. By the middle of the 20th century it became clear that DNA and RNA were carriers of hereditary information; then their structure was described, and a little later numerous methods appeared that allowed these molecules to be "cut" in vitro using cellular enzymes - restrictases [2]; amplify them using PCR [3]; and even read the sequence of specific genes and genomes using multiple sequencing methods [4].
Initially, work with genetic material was performed literally "in the kitchen" and could not always be reproduced even in the neighboring scientific laboratory. But time passed, approaches changed, and methods were standardized to such an extent that they were gradually introduced into applied research and even biotechnological production.
In 1984, the scientific community was stirred by the news that scientists managed to isolate and read a fragment of DNA of the Burchell zebra, which is extinct and survives only in museum collections [5]. A year later, Swedish geneticist Svante Pääbo confirmed the possibility of using museum and archaeological material for fundamental scientific research, having first analyzed the genetic material of Egyptian mummies. Years later, it turned out that the samples he analyzed were contaminated with modern genetic material [6], but the development of sequencing methods still made it possible to work with even minimal amounts of poorly preserved DNA.
Forensic scientists were also interested in the new techniques of genetic material analysis. The fact is that the methods of classical dactyloscopy and blood group analysis, which were customary at that time, had their limitations, and in some cases they misfired.
In 1984, the British scientist Sir Alec John Jeffreys (Figure 3) developed and presented a method of identifying a person using his genetic material. Later this approach was called DNA identification, winning love and respect of criminologists all over the world. Jeffreys was knighted for his work.
A bit of history or what does it have to do with knights?
150 years ago Johannes Friedrich Miescher discovered nucleic acids, a concept that eventually turned the world upside down [1]. By the middle of the 20th century it became clear that DNA and RNA were carriers of hereditary information; then their structure was described, and a little later numerous methods appeared that allowed these molecules to be "cut" in vitro using cellular enzymes - restrictases [2]; amplify them using PCR [3]; and even read the sequence of specific genes and genomes using multiple sequencing methods [4].
Initially, work with genetic material was performed literally "in the kitchen" and could not always be reproduced even in the neighboring scientific laboratory. But time passed, approaches changed, and methods were standardized to such an extent that they were gradually introduced into applied research and even biotechnological production.
In 1984, the scientific community was stirred by the news that scientists managed to isolate and read a fragment of DNA of the Burchell zebra, which is extinct and survives only in museum collections [5]. A year later, Swedish geneticist Svante Pääbo confirmed the possibility of using museum and archaeological material for fundamental scientific research, having first analyzed the genetic material of Egyptian mummies. Years later, it turned out that the samples he analyzed were contaminated with modern genetic material [6], but the development of sequencing methods still made it possible to work with even minimal amounts of poorly preserved DNA.
Forensic scientists were also interested in the new techniques of genetic material analysis. The fact is that the methods of classical dactyloscopy and blood group analysis, which were customary at that time, had their limitations, and in some cases they misfired.
In 1984, the British scientist Sir Alec John Jeffreys (Figure 3) developed and presented a method of identifying a person using his genetic material. Later this approach was called DNA identification, winning love and respect of criminologists all over the world. Jeffreys was knighted for his work.
DNA dactyloscopy: advantages and disadvantages
Genetic analysis of biological samples obtained at crime scenes has greatly simplified the work of investigators. They now have a reliable tool for identifying the perpetrator or victim, obtaining incontrovertible evidence and solving crimes.
The main advantages of genetic fingerprinting are the ability to work even with small amounts of biological material and the high accuracy that allows for identification of an individual - if all the requirements for analysis are met, its reliability exceeds 99%. This approach has become one of the most important in the investigation of crimes [7].
It is important to note that classical DNA dactyloscopy methods do not allow identifying identical twins whose genetic profile is the same, since they are formed from the same fertilized egg.
For DNA analysis, first of all, DnA itself is needed, but it is far from always possible to extract high-quality genetic material from biological samples that have been exposed to chemical and thermal factors. Once in the environment, this molecule enters into chemical reactions, is destroyed (fragmented) and modified, although in favorable conditions it can persist for thousands of years [8].
It is known that the most ancient genetic material of human ancestors was extracted from the bone remains of our humanoid relatives who lived on the territory of the Iberian Peninsula (Sierra de Atapuerca) about 430 thousand years ago [9]. The specific microclimate of some caves with low humidity and temperature values significantly increases the lifetime of the genetic material. However, DNA in the samples found at the crime scene is often represented in minimal quantities and, as in archaeological samples, is severely degraded.
In Europe and the United States, attention is drawn to another disadvantage of genetic fingerprinting, related to interference with a person's privacy and violation of privacy.
Human rights activists fear that the genetic information of criminals and criminal suspects could fall into the hands of third parties and then be misused [10]. For example, there are already known cases of the use of genetic information to control Muslim minorities in the Chinese province of Xinjiang.
The U.S. is also planning to systematically collect DNA profiles of immigrants in federal custody [11]. Obviously, the fears of human rights activists are not groundless and have real grounds.
The main advantages of genetic fingerprinting are the ability to work even with small amounts of biological material and the high accuracy that allows for identification of an individual - if all the requirements for analysis are met, its reliability exceeds 99%. This approach has become one of the most important in the investigation of crimes [7].
It is important to note that classical DNA dactyloscopy methods do not allow identifying identical twins whose genetic profile is the same, since they are formed from the same fertilized egg.
For DNA analysis, first of all, DnA itself is needed, but it is far from always possible to extract high-quality genetic material from biological samples that have been exposed to chemical and thermal factors. Once in the environment, this molecule enters into chemical reactions, is destroyed (fragmented) and modified, although in favorable conditions it can persist for thousands of years [8].
It is known that the most ancient genetic material of human ancestors was extracted from the bone remains of our humanoid relatives who lived on the territory of the Iberian Peninsula (Sierra de Atapuerca) about 430 thousand years ago [9]. The specific microclimate of some caves with low humidity and temperature values significantly increases the lifetime of the genetic material. However, DNA in the samples found at the crime scene is often represented in minimal quantities and, as in archaeological samples, is severely degraded.
In Europe and the United States, attention is drawn to another disadvantage of genetic fingerprinting, related to interference with a person's privacy and violation of privacy.
Human rights activists fear that the genetic information of criminals and criminal suspects could fall into the hands of third parties and then be misused [10]. For example, there are already known cases of the use of genetic information to control Muslim minorities in the Chinese province of Xinjiang.
The U.S. is also planning to systematically collect DNA profiles of immigrants in federal custody [11]. Obviously, the fears of human rights activists are not groundless and have real grounds.
How it works
DNA dactyloscopy methods are based on human genetic variability. Nucleotide substitutions in DNA (depending on the frequency in the population, they are also called DNA polymorphisms, or mutations) make us individually different. And these differences can be found in both nuclear and mitochondrial genomes, small circular DNA molecules that regulate the functioning of mitochondria, the energy "factories" of cells.
It should be noted that DNA polymorphisms can be found in the genome of each of us - they become our unique DNA barcode. Some of them can cause serious diseases [12], but in most cases they have no effect on our vital functions.
Expert Commentary: «The use of genetic analysis helps solve many crimes: thefts, kidnappings, and murders. There are cases when the use of molecular-genetic methods allowed the identification of a criminal in an unsolved case or the release of an innocent person. One such example is the investigation of the rape of a girl in Illinois in 2000. Genetic analysis of biological material left as a result of the crime would have helped identify the perpetrator, but at that time the investigation did not have the necessary equipment. Circumstantial evidence pointed to a man who for a long time was listed as a suspect, but was never convicted. In 2005 the DNA-analysis laboratory was able to conduct genetic analysis of the preserved biological samples. The results showed that the man who had been suspected of the crime all those years was innocent, and the DNA material found belonged to a 44-year-old man who had already served time in one of the state prisons.
But sometimes work with genetic material can lead to unexpected results and even bring the investigation to a dead end. An example of such a case is the criminal investigation into the kidnapping and murder of a young boy, on whose clothes was found the blood of an unidentified man. The investigation conducted a genetic analysis of the blood sample, but its comparison to the genetic profiles of more than 2,000 people showed no match. It turned out that the blood traces found belonged to the investigator who conducted the inspection of the scene. Despite the fact that he was wearing gloves, there was an accidental transfer of his genetic material: he killed a mosquito and thus transferred the "trace" of his blood to the clothes of the corpse during the seizure of items.
Another example where genetic analysis misled investigators is the famous case of the «Heilbronner Phantom» (Woman Without a Face). During the investigation of a wide variety of unrelated cases (robberies, burglaries, murders, etc.) in various countries (Austria, Germany, and France), the DNA of the same woman was regularly found at the crime scene. It was later determined that the carrier of the detected DNA was a packer in a cotton swab factory, which the forensic specialists used to collect physical evidence. The cotton swabs were accompanied by a specification stating that they were sterile (i.e., free of live microorganisms), but there was no information that they were not contaminated with foreign DNA and could be used to collect forensic samples».
Modern DNA dactyloscopy methods use a variety of genetic variants in different regions of the genome. For example, analysis of tandem repeats - short repetitive genome sequences whose number differs in two randomly sampled individuals [13] - allows a clear paternity test or finding additional evidence against a criminal suspect.
Y-chromosomal DNA markers can be used to determine a person's sex. Genetic markers provide information on the ethnicity of an individual's relatives, eye color or hair color.
Moreover, epigenetic information (e.g., DNA methylation) makes it possible to estimate the biological age of individual cells, tissues, and the organism as a whole [14]. In this article I will talk more about the above methods of genetic analysis. And the use of epigenetic methods in the drug business will be the subject of my next publication.
Y-chromosomal DNA markers can be used to determine a person's sex. Genetic markers provide information on the ethnicity of an individual's relatives, eye color or hair color.
Moreover, epigenetic information (e.g., DNA methylation) makes it possible to estimate the biological age of individual cells, tissues, and the organism as a whole [14]. In this article I will talk more about the above methods of genetic analysis. And the use of epigenetic methods in the drug business will be the subject of my next publication.
DNA collection and isolation from biological material
DNA extraction and analysis from forensic samples is, at first glance, comparable to art. Sometimes it is impossible to imagine that a few drops of blood or a piece of skin under a victim's fingernails can become the most important evidence in a criminal investigation.
In fact, the actions of crime scene forensic specialists are fine-tuned and follow a single scenario, one of the main goals of which is to find biological material suitable for DNA extraction. Any biological tissue and human secretions can be used for this purpose:
- Bone remains
- Teeth
- Hair follicles
- Blood
- Epithelial cells
- Sweat
- Saliva
- Sperm samples
- Excrement, etc.
Biomaterial in the evidence warehouse can last for decades. Often, the examiner will only take a portion of the material for examination, as much as he needs for DNA extraction. For example, if there are traces of blood on the knife, the expert does not wash off all the blood, but makes a small rinse just before the DNA extraction. This leaves traces on the knife, which can be used to conduct a re-examination several decades later.
In the cell nucleus, DNA is in close contact with numerous organic molecules necessary for its effective functioning. However, for genetic analysis, carbohydrates, lipids, and proteins must be removed from the test sample, which can reduce the efficiency of the methods used and, consequently, affect the solvability of crimes.DNA extraction takes little time thanks to a variety of commercial kits and systems specifically designed for nucleic acid extraction (e.g., the AutoMate Express DNA Extraction System from Thermo Fisher Scientific). In addition, in large forensic centers with thousands of analyses per day, this process is fully automated and carried out with the participation of robots under the supervision of an operator.
By the way, robotic systems are widely used not only in forensic laboratories, but also in many medical and biotechnology centers, allowing to speed up the process, reduce the cost of work and significantly reduce the chance of error.
After extraction, the DNA is dissolved in a special compound, where it can be stored for years (at -20°C or -80°C) if necessary.
Genetic analysis
Immediately after DnA extraction, a specialist is faced with the question of further ways to analyze it. Since the introduction of DNA identification in forensics, molecular biology techniques have changed so much that there are several possible variants. Our further story will highlight the various techniques of DNA analysis that forensic experts use in their practice.
Restriction fragment length polymorphism (RFLP analysis)
RFLP analysis is historically one of the first methods for DNA identification. It is based on the use of special cellular enzymes, restrictases. Restriction enzymes can recognize certain sites on a nucleic acid molecule and cut it through these sites. Several thousand such enzymes have been described to date. After cutting the DnA molecule with restriction enzymes, the lengths of the obtained fragments are evaluated using gel electrophoresis (Figure 9).
Immediately after DnA extraction, a specialist is faced with the question of further ways to analyze it. Since the introduction of DNA identification in forensics, molecular biology techniques have changed so much that there are several possible variants. Our further story will highlight the various techniques of DNA analysis that forensic experts use in their practice.
Restriction fragment length polymorphism (RFLP analysis)
RFLP analysis is historically one of the first methods for DNA identification. It is based on the use of special cellular enzymes, restrictases. Restriction enzymes can recognize certain sites on a nucleic acid molecule and cut it through these sites. Several thousand such enzymes have been described to date. After cutting the DnA molecule with restriction enzymes, the lengths of the obtained fragments are evaluated using gel electrophoresis (Figure 9).
Since there are no completely identical human genomes (except for identical twins), the difference or similarity in the resulting DNA fragment length profiles can be a good marker for both personal identification and kinship determination.
However, this method involves DNA fragmentation, and is therefore sensitive to the quality and quantity of the original genetic material. That is why the RFLP analysis is not practically used at present - unlike other methods, which will be discussed below.
Analysis of the number of tandem repeats in the genome
Tandem repeats are copies of the same short DnA sequence repeated one after another [15]. Repeats that are of interest to forensic scientists include loci with a varying number of tandem repeats (VNTR) and short tandem repeats (STR). They are also called minisatellites and microsatellites, respectively.
The essence of this method is the PCR amplification of DNA fragments containing these short repetitive sequences whose lengths are different in two randomly sampled individuals. After amplification, the length of the obtained fragments is evaluated using gel electrophoresis or capillary electrophoresis.
The Quantifiler DNA Quantification Kit and the QuantStudio system from Termo Fisher Scientific can be used for this purpose. The QuantStudio is a compact benchtop instrument with a wide range of features.
As with the previous method, the main identifying factor of the STR assay is the length of the obtained fragments, which is unique for each individual and depends on the number of repeats in the analyzed site. STR-analysis is characterized by high accuracy and speed, as well as low cost. The quality of genetic material is an important condition for the analysis.
However, this method involves DNA fragmentation, and is therefore sensitive to the quality and quantity of the original genetic material. That is why the RFLP analysis is not practically used at present - unlike other methods, which will be discussed below.
Analysis of the number of tandem repeats in the genome
Tandem repeats are copies of the same short DnA sequence repeated one after another [15]. Repeats that are of interest to forensic scientists include loci with a varying number of tandem repeats (VNTR) and short tandem repeats (STR). They are also called minisatellites and microsatellites, respectively.
The essence of this method is the PCR amplification of DNA fragments containing these short repetitive sequences whose lengths are different in two randomly sampled individuals. After amplification, the length of the obtained fragments is evaluated using gel electrophoresis or capillary electrophoresis.
The Quantifiler DNA Quantification Kit and the QuantStudio system from Termo Fisher Scientific can be used for this purpose. The QuantStudio is a compact benchtop instrument with a wide range of features.
As with the previous method, the main identifying factor of the STR assay is the length of the obtained fragments, which is unique for each individual and depends on the number of repeats in the analyzed site. STR-analysis is characterized by high accuracy and speed, as well as low cost. The quality of genetic material is an important condition for the analysis.
STR analysis appeared in practical forensics almost twenty years ago, but to this day remains the main method of identifying individuals. The results of DNA analysis of suspects and criminals - genetic profiles - are entered by criminologists into special databases.
Therefore, when biological traces are found at crime scenes from which DNA can be isolated, it has become possible to identify all people who have already come to the attention of law enforcement agencies. The same markers are used to search for missing persons and to establish paternity.
Therefore, when biological traces are found at crime scenes from which DNA can be isolated, it has become possible to identify all people who have already come to the attention of law enforcement agencies. The same markers are used to search for missing persons and to establish paternity.
As a rule, systems designed to identify individuals allow the analysis of several genome loci (10-24) at once, including the amelogenin protein gene, by which sex is determined. The amelogenin gene is found on both the X and the Y chromosome, but differs in size, which allows the sex of a person to be determined.
Thanks to the use of high-throughput sequencing technologies, forensics has obtained an effective tool that has opened up new opportunities for simultaneous analysis of multiple sites (loci) of the nuclear and mitochondrial genomes.
An important advantage of these methods is the ability to distinguish even identical twins (by somatic mutations), which is impossible with RFLP- or STR-analysis.
Thanks to the use of high-throughput sequencing technologies, forensics has obtained an effective tool that has opened up new opportunities for simultaneous analysis of multiple sites (loci) of the nuclear and mitochondrial genomes.
An important advantage of these methods is the ability to distinguish even identical twins (by somatic mutations), which is impossible with RFLP- or STR-analysis.
The second part of this publication will describe how the methods described above can be used to identify drug labs and drug dealers, and how you can confuse forensics by cleaning up your tracks.
Read PART II
