The DNA department of the Estonian Forensic Science Institute (EKEI) operates labs in both Tallinn, at EKEI's headquarters, and Tartu, within the Biomedicum facility. According to department head Maarja Sadam, the team's work is divided into three main areas.

"The first and most important task is conducting DNA analyses for criminal investigations on behalf of the police. Each year, we perform approximately 1,300 expert examinations and analyze around 8,000 samples from crime scenes," Sadam explains.

The department's second key responsibility is managing the National Biometric Registry for Criminal Proceedings (RSBR), a DNA database related to criminal activity. This work includes an international dimension through the PRÜM data exchange agreement, which facilitates DNA data comparisons with most European Union countries.

"Our third area of focus is determining biological relationships, a service we provide both to courts and private individuals," Sadam adds.

From cotton swabs to blades of grass

When a crime is committed, the police collect all potentially relevant items from the scene and send them to the Estonian Forensic Science Institute. Before other departments begin their analysis, the DNA department takes the lead in identifying genetic material on the items.

"These could include biological traces on clothing. Are they bloodstains? Is there semen? Whose DNA is it? These are the most common questions we address," Sadam explains.

The number of items sent from a single crime scene can range from one to hundreds. "The most typical submissions are swabs, which the police have already used to collect DNA samples," Sadam notes. Other commonly analyzed objects include clothing, packaging for narcotic substances, axes, knives and screwdrivers.

Unusual cases also arise. For instance, laboratory submissions may include a blade of grass with traces of semen or urine marks left on snow. "Such evidence is transported in a cooler box and requires storage at sub-zero temperatures," adds DNA specialist Silja Erg.

The DNA department examines the items and extracts DNA samples. According to Maarja Sadam, their work frequently involves analyzing traces of blood, saliva and semen. "The largest category of cases involves contact traces left when a person touches a surface. Our focus is not on fingerprints themselves but on the cells left behind on the surface," Sadam clarifies.

Once samples are collected and analyzed, they are compared against the DNA profiles of all suspects. The police collect DNA samples from suspects and, if necessary, from victims as well. While the initial lab analysis takes about two days, a complete expert examination typically requires around three weeks. "We don't run just one reaction on a single sample. Instead, we perform two to four reactions, which naturally takes time," Sadam explains.

In routine analyses, experts examine 16 DNA markers along with one marker indicating biological sex. "Most of the markers used in daily comparisons carry no meaningful information about external traits. They are specifically selected for personal identification, allowing us to distinguish individuals and determine the donor of biological material," says Silja Erg.

When there is a need to determine traits like eye, hair or skin color, or a person's biogeographical ancestry, a more advanced analysis is performed. This involves comparing a broader set of specialized markers.

At EKEI, all DNA samples are translated into numerical data, enabling experts to compare numbers marker by marker in search of matches. "For a quality DNA profile from a single individual to link them to a suspect, it must match 100 percent," Sadam explains. However, mixed DNA profiles containing material from multiple individuals present a greater challenge. In such cases, experts can determine the proportion of DNA contributed by each donor, identifying who contributed more and who contributed less.

While forensic analysis can establish a connection between a sample or object and an individual, Sadam emphasizes that further interpretation lies with the police and prosecutors. "It doesn't work in a way where the person leaving the most DNA is automatically the perpetrator," says Silja Erg. "Ultimately, it is the court that determines guilt."

The color of crime

Analyzing samples from crime scenes traditionally relied on microscopes and the trained eyes of forensic experts. However, EKEI's DNA labs now utilize cutting-edge technology to speed up these processes. One such innovation is a lighting device that has been in use for a few years, making invisible traces visible.

"For example, saliva stains or blood on black fabric can't be seen with the naked eye, but specific wavelengths of light make them visible," Maarja Sadam says.

Housed in a small, windowless room divided into "clean" and "dirty" areas, the lighting device requires meticulous operating procedures. Silja Erg explains that two people work together – one handling samples in the clean area while the other inputs data in the dirty area, ensuring no contamination occurs. "Every part of the body must be covered. In this room, you can't breathe or sneeze on anything," Erg points out with a smile.

To demonstrate the device, Erg brings several garments from a teaching collection: a pair of jeans and two brightly colored shirts. The analysis begins with a visual inspection for biological stains. "For example, if these were the victim's jeans, we would check for logical sampling spots – like the leg area if the victim had been dragged by their legs," Erg explains.

If stains are not visible, the lighting device comes into play. The device features a range of colored lamps and filters that can highlight different materials, making molecules fluoresce while filtering out background noise. "The choice of light and filter depends on the fabric and stain. Blue UV light, familiar from films, works well for saliva on jeans, while dried blood on a colorful shirt might require the infrared spectrum," Erg elaborates.

Once a stain is identified, the expert collects a sample using a swab, which is then stored in a special tube. The expert decides whether to immediately extract DNA from the sample or conduct further microscopic analysis. This step is particularly relevant in rape cases, where identifying sperm cells is crucial. For this, cellular material from the sample is stained using a method known as the "Christmas tree technique."

"This technique stains the nucleus of a sperm cell red and the cytoplasm green, resembling ornaments on a Christmas tree," Sadam describes.

Following staining, sperm cells are analyzed in a separate lab equipped with an AI-based microscope system, currently being validated by Erg and expected to be fully operational early next year. The system promises to speed up investigations of rape cases by detecting sperm even in samples with significant background noise.

"Forensic samples from crime scenes are often harder to analyze because they contain more than just cells," Erg notes. "This is where AI helps significantly, as it can find sperm amidst all the background material."

The system begins by determining the best focal plane for imaging, given the microscopic size of cells. It then differentiates objects on the slide – such as cells, dust particles and bacteria – and photographs each one. The AI compares the images against a database of sperm-specific characteristics.

"It then calculates how similar each object is to sperm, with results ranging from 0 to 100 percent. Any positive findings are always confirmed by a human expert," Erg explains.

The AI system is especially valuable for analyzing large items like bedsheets. Previously, multiple random samples would need to be taken from such items. Now, the technology highlights the precise areas to investigate, making the process faster and more effective.

"Our work has become quicker and more efficient," Erg concludes, underscoring the transformative impact of advanced forensic tools in solving complex cases.

Kinship can now be determined at the level of cousins

DNA profiles collected from suspects for forensic purposes are entered into RSBR. "Currently, our registry contains about 70,000 individual profiles, representing approximately 5.4 percent of our population," says Maarja Sadam. Additionally, the database includes roughly 10,000 unidentified crime scene profiles, which have not been matched to any individual. Around 5,000 new profiles are added annually.

"According to forensic law, individual profiles are retained in the registry for 40 years, while crime scene traces are stored for 75 years," Sadam explains. DNA samples collected from individuals who were present at a scene by chance or for exclusion purposes are not stored long-term. Crime scene profiles, however, are shared with European countries under the PRÜM agreement to facilitate cross-border investigations if necessary.

In the department's third area of activity – kinship determination – a significant technological leap has been made. "Before 2024, our methodology allowed us to establish only first- and second-degree relationships," Sadam notes. This meant identifying relationships such as parent-child or grandparent-grandchild connections, as well as biological uncles or aunts. "Our dream was to go much further and determine relationships up to first cousins," Sadam recalls.

In 2022 and 2023, the DNA department participated in an international collaboration project to test a marker set called FORCE, developed by researchers in the United States and Sweden. This set, containing approximately 5,400 markers, enables the identification of more distant kinship relationships. After successful trials and validation at the DNA department, the method was officially adopted at the beginning of 2024.

"We can now determine third- and fourth-degree relationships, such as first cousins or half-first cousins," Sadam explains. In certain cases, the analysis can go even further, identifying relationships with first cousins' children. "Essentially, anyone seeking to confirm paternity or other types of lineage can now use this service," Sadam adds.

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