DNA Profiling: Tracing Killers and Solving Mysteries Using Genetic Clues

I

n the mid-1980s, tragedy struck two villages in Leicestershire, England. In two separate instances, three years apart, two teenage girls were raped and murdered on their way home from school. On investigating the commonalities between the two cases, the police suspected 17-year-old Richard Buckland, as the serial killer behind these crimes.

Although the police were convinced that Buckland was responsible for both murders, his inconsistent testimony and lack of other hard evidence left them at a dead end. Luckily, around the same time, geneticist Sir Alec Jeffreys of the University of Leicester had successfully developed a new DNA profiling method, originally intended for paternity testing. While few doubted that this technique could be used to apprehend criminals, the police believed it could conclusively link Buckland to both murders. The investigators teamed up with Jeffreys to analyze the DNA samples found at both crime scenes; the DNA fingerprint method not only helped them crack the case, but it also revolutionized the field of forensics.

The Birth of DNA Profiling: Can it Catch a Killer?

DNA is the blueprint for inherited traits like hair, eye color, and blood type. But just as no two fingerprints are the same, Jeffreys and other researchers investigated the concept of unique DNA patterns that could be used to identify individuals. In the early 1980s, genetic typing was in its infancy, with researchers using restriction fragment length polymorphisms (RFLPs) to detect variations in DNA sequences.¹ This method, which employed single locus probes to target specific regions, provided limited information about an individual’s genetic makeup. However, Jeffreys sought to develop more robust and informative genetic markers to aid in identification.

As Jeffreys homed in on studying DNA variation between individuals, he and his colleagues discovered short tandem repeat (STR) DNA sequences, known as minisatellite regions, scattered throughout the genome.² Within these segments, they found a shared short sequence that revealed highly variable tandem repeats, which seemed ideal for unique genetic profiling.

By combining RFLP and STR analysis, scientists could slice DNA with a restriction enzyme, sort the fragments by size, and transfer them to a Southern blot for analysis. However, early experiments still did not give him a clear picture of these repeated regions.

In 1984, Jeffreys had a fortunate breakthrough when he analyzed a DNA sample from his lab technician, her parents, and nonhuman sources. Using probes to target minisatellite regions, he produced a barcode-like image with distinct patterns specific to each individual. It was the first ever DNA fingerprint, which opened a whole new world of forensic possibilities.³

Shortly after, in collaboration with Peter Gill from the Forensic Science Services (FSS), who developed extraction methods to separate sperm from vaginal fluid—a method that is still used today—performed the first forensic application for DNA profiles from blood and semen stains.⁴

So, when the police contacted Jeffreys to help with the Leicestershire cases, Jeffreys applied this technology to test if Buckland's DNA from his semen matched the samples found at the crime scenes. To everyone’s surprise, Buckland was not a match.

“When Alec Jefferys first came up with this result [for the suspect], the police didn’t believe him, so [the FSS] had to get involved to verify it,” said Gill, now at the University of Oslo.

But while Buckland was exonerated, the investigators still needed to find the true killer, who remained at large. They decided to use the same technology to try to catch the killer. In early 1987, the local authorities and the FSS conducted a DNA-based manhunt for men between the ages of 16 to 34 to voluntarily give blood samples for DNA testing. They were looking for someone who had type A blood. Thousands of samples flooded into FSS’s research facility in Aldermaston during the UK's first mass DNA screening. Out of 5,000 men tested, 4,000 passed blood ABO profiling, and 1,000 underwent DNA fingerprinting: Yet none matched the crime scene samples.

As only a handful of people could perform this assay, Gill often joined casework efforts. “Basically, the difficulty was that none of this had been validated, so they relied on our laboratory, and there was a lot of pressure,” Gill recalled. Over six grueling months, he worked weekends processing samples, each of which took two weeks to complete. Despite these efforts, no match surfaced.

A breakthrough came a year later when someone overheard a pub conversation where a man admitted he’d been paid to submit fake samples. Soon, the real culprit, Colin Pitchfork, was tested and found to be a match. His DNA linked him to both teen murders and other crimes. Pitchfork became the first person convicted through DNA evidence, a landmark case that reshaped forensics.

“DNA profiling laid the groundwork and the thoughts behind the national DNA database because people realized that it wasn't so much the finding of the suspect, but the elimination of all of the 5,000 individuals,” remarked Gill. This nascent technology sparked significant interest in DNA’s potential as a forensic tool, captivating scientists and law enforcement and inspiring Joseph Wambaugh’s book The Blooding, which detailed the twists and turns of the case.

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How PCR Amplified the Impact of DNA Profiling in Forensics

John Butler, now a forensic scientist at the National Institute of Standards and Technology (NIST), vividly remembered reading The Blooding in the summer of 1990, when he studied chemistry as an undergraduate student at Brigham Young University. This book’s account of using DNA to solve a crime in England piqued his interest in forensic science.

At the time, DNA profiling was gaining traction, but the process with RFLP remained slow, tedious, and required substantially larger sample sizes. When polymerase chain reaction (PCR) technology entered the scene in the mid-1980s, DNA amplification got easier with smaller sample size and time investment requirements.⁵ Soon, researchers used fluorescent probes for real-time amplification detection.⁶

During graduate school at the University of Virginia, Butler’s research, facilitated by his advisor’s connection with the Federal Bureau of Investigation (FBI) lab, worked on refining DNA testing techniques. In the early 1990s, the FBI had begun exploring STR markers, whose short, two- to six-base pair sequences made them more efficient with PCR, which provided faster and more reliable amplification than RFLP.⁷

At the same time, across the pond, Jeffreys used STR markers in tandem with PCR, while Gill and his colleagues further refined this process to detect multiple loci.^8,9 While RFLP and PCR used slab gels to separate DNA fragments, Butler developed capillary electrophoresis (CE) to speed up and automate DNA band separation, which also enabled higher resolution and smaller DNA fragment detection.¹⁰ “The methods I worked on are now used worldwide. Every single lab in the world uses capillary electrophoresis for DNA testing. So, it was kind of neat to be part of that very first effort with some of the very first STR markers.”

Tracing Ancestry Using DNA Profiling: How STRs Solved a Century-Old Mystery

Beyond criminal and paternity investigations, DNA profiling also helped solve a historical mystery in determining the identity of Russia’s last Imperial family, the Romanovs. In 1918, Tsar Nicholas II, Tsarina Alexandra, and their children—Olga, Tatiana, Maria, Anastasia, and Alexei—were executed. However, the grisly details and location of their final resting place remained a secret for nearly a century. In 1991, nine skeletal remains, believed to be of the Romanov family, were excavated from a mass grave. Researchers wondered if DNA profiling would work on these samples. Russian geneticist Pavel Ivanov from the Engelhardt Institute of Molecular Biology enlisted the aid of British DNA experts and reached out to Gill, who agreed to the challenge.

[In the Romanov case], that was the first time we used them short tandem repeats, moving away from the RFLP method.

—Peter Gill, University of Oslo

“He came over to the UK clutching these bones in a sort of supermarket bag. I met him at the airport, and we went off to carry out this work, which took about a year. It was actually quite complex for various reasons,” said Gill.

“[In the Romanov case], that was the first time we used them short tandem repeats, moving away from the RFLP method,” said Gill. “It demonstrated that we could use these fragments to analyze very degraded samples. These were 70-year-old samples of bones that had been in the ground, and we were able to get profiles from them.” The researchers took small sections of the bones for analysis. As expected, they only found tiny amounts of DNA present in the bones, from just half a dozen cells.

Using autosomal STRs, they determined that five of the nine skeletons were related—a mother, a father, and three daughters. But were these truly the remains of the Romanov family? To confirm their identities, researchers turned to mitochondrial DNA (mtDNA), which passes unchanged from mother to child.

They obtained blood samples from Prince Philip, Duke of Edinburgh, who was a direct descendant of the Tsarina Alexandra, and it was a match; this was the Tsarina Alexandra.¹¹ The children’s samples also matched, wherein the researchers identified two daughters as Olga and Tatiana; however, the third daughter could have been either Maria or Anastasia. Then, to confirm the Tsar’s identity, they found two distant maternal relatives that also matched, but except at a single position: a heteroplasmy. This tiny mismatch fueled controversy.

To confirm the authenticity of the heteroplasmy, another team at the Armed Forces DNA Identification Laboratory (AFDIL) tackled this identity mystery with the exhumed remains of Grand Duke Georgij Romanov, the brother of Tsar Nicholas II, with mtDNA. Mitchell Holland, who worked on this case at AFDIL and is now at Pennsylvania State University (Penn State), said, “There had been testing that had already been done, but our testing kind of gave the last pieces of information that allowed them to move forward with an identification.” Their mtDNA matched down to the heteroplasmy at the same position, proving their relationship. This was the last Tsar.¹² “So, it was a real interesting case from that scientific DNA perspective.”

But what of the remaining missing children, Alexei and one of the youngest daughters, Maria or Anastasia? Did they suffer the same fate, or did they manage to escape? One of the most famous claimants was Anna Anderson, who insisted that she was Anastasia for decades even until her death in 1984. A postmortem DNA test was conducted on her hair and a tissue sample was recovered from a Virginia hospital through a collaboration between FSS, AFDIL, and Penn State. Gill recalled traveling to Virginia to retrieve the tissue sample, which he described as, “a bit of her bowel which had been preserved in paraffin wax.” He wasn’t sure if this sample would work, but the lab was able to process it. Anderson’s mtDNA did not match the Romanovs or their living relatives.¹³

In 2007, researchers discovered additional remains—bone fragments and teeth—near the 1991 grave. Could these belong to the last two members of the Romanov family? Researchers used mtDNA to make comparisons with DNA from earlier work, autosomal STR testing to confirm the parental relationship between these remains and those of Tsar Nicholas II and Alexandra, and Y chromosome STR testing from a living Romanov cousin. The DNA analysis from all three tests confirmed the identity of the samples and accounted for the entire Romanov family.¹⁴

“The Romanov case and the Waco case, led to the universal acceptance of short tandem repeats,” said Gill, where forensic scientists had to contend with old bones and burned samples with highly degraded DNA from victims of the mass disaster near Waco, Texas.¹⁵

STRs in the System: How the CODIS Database Reshaped Forensics

In 1995, the FSS launched the world’s first national DNA database to track STR markers. Recognizing their potential, other countries soon followed suit. Around this time, Butler had a similar idea when he went to NIST as a postdoctoral researcher.

“I collected all this stuff [on STR markers] and then gathered it in a way that would be useful to other people. I saw STRs were just starting to take off,” said Butler. He initially intended to compile his findings into a review article. However, with the growing availability of the World Wide Web, he saw an opportunity to create a dynamic resource. “I thought, ‘let’s see if I can figure out how to code stuff in HTML and make this a living system,’” he recalled. The result was STRbase, launched in early 1997, a comprehensive database that has since become an essential tool for forensic scientists worldwide.

Then, the US developed the Combined DNA Index System (CODIS) in 1998 to aid law enforcement in integrating DNA in their investigations. CODIS initially selected 13 core STR loci, though only eight overlapped with the UK’s FSS database. This lack of alignment posed challenges for international comparisons until 2017 when CODIS added seven more markers to match its European counterpart.

Even as forensics researchers worked to advance next-generation DNA testing systems, mass fatality incidents, such as the World Trade Center attack in 2001, posed unprecedented difficulties.

“The temperature at ‘Ground Zero’ was upwards of 1,000 to 2,000 degrees. It was a crematorium of sorts,” said Holland. He also joined efforts in processing more than 13,000 skeletal fragments. “Those were the most challenging samples we ever worked on. A bone sitting in a grave site will have a decent amount of DNA, but those bones [from Ground Zero] were in terrible shape.” These conditions left very limited DNA information—if any at all. Traditional STR markers struggled to produce results from degraded samples, prompting efforts to develop smaller DNA fragments.

“There were efforts that we did at NIST and in collaboration with others to make ‘miniSTRs’ that led to them shrinking the size of the test. We’re looking at smaller fragments of DNA and recovering information from that,” said Butler. These miniSTRs, along with additional analyses such as single nucleotide polymorphisms (SNPs) and mtDNA enabled researchers to process highly degraded samples.¹⁶ Roughly 40 percent of victims have yet to be identified, and researchers have quietly been employing their expanding DNA analyses to bring closure to grieving families.

More recently, forensic investigative genetic genealogy has garnered interest. This technique involves uploading a crime scene DNA profile to public genealogy databases, such as Ancestry, 23andMe, and MyHeritage, which use SNP analysis to detect genetic matches. Instead of identifying the suspect directly, they trace DNA connections through family trees, pinpointing relatives to narrow down potential offenders. The technique made headlines in 2018 when it led to the identification of the Golden State Killer, marking the first major success of this approach in solving a decades-old case.

In addition to SNP analysis, efforts are underway to build comprehensive Y chromosome STR and mtDNA databases.¹⁷ “We started out with having less than 10 markers that we could work with, and now there are thousands of SNP markers that can be done on the Y chromosome and several dozen well-characterized STR markers,” remarked Butler.

Holland and his team have started sequencing 10,000 mitochondrial genomes to support this advancement. “If the community moves forward with this kind of testing, they’ll need a database to compare the DNA profile against and calculate how often that profile might appear in the population,” Holland explained. Since the odds of two individuals sharing the same mtDNA profile are as low as one in tens or hundreds of thousands, creating a large reference database is essential for accurate comparisons and statistical analysis.¹⁸

Beyond Profiling: The Next Frontier in Forensic DNA Analysis

Although DNA analysis has become a staple in forensic science, the public’s fascination with true crime, courtroom dramas, and TV shows has morphed into a bias that many call the “CSI effect.” While DNA evidence is powerful, it does not provide a definitive answer. Crime scene samples rarely arrive in pristine condition. Analysts often work with degraded or mixed samples from multiple contributors, which makes interpretation far more complex than what TV shows portray. Even with a clear profile, DNA alone doesn’t solve the case—it’s all about context.

There's a lot more nuances with DNA testing than people appreciate.

—John Butler, National Institute of Standards and Technology

“It's no longer just a simple question of who left that sample, but how was it left, and what were the activities involved in leaving it?” said Butler.

Advances in DNA testing have made it possible to detect trace amounts of genetic material, but this sensitivity comes with challenges, said Gill. Imagine swabbing a room for house dust, which is packed with DNA. People shed millions of skin cells daily, leaving traces behind just by passing through. While it reveals who’s been in space, it’s far from incriminating evidence. “There's a lot more nuances with DNA testing than people appreciate, and that's the hard part to communicate because you have to overcome that barrier of people thinking DNA solves everything,” said Butler.

This emerging use of environmental DNA (eDNA) in forensic casework offers new possibilities.¹⁹ By swabbing door frames or filtering the air in a room, investigators can gather a historical record of who has been in a space, potentially generating leads. It’s a tool for intelligence purposes, not incrimination, Gill explained. For example, an air filtration device could reveal who was recently present in a room, helping investigators focus their efforts. However, such evidence must be used alongside other investigative work—it’s a lead, not a conclusion.

Advances in DNA testing, including eDNA, continue to push forensic science forward, but they also raise new challenges in how evidence is interpreted and communicated to the public. Researchers remain dedicated to refining these methods to ensure DNA remains a valuable tool in solving crimes without being seen as a definitive answer on its own.

With ongoing progress and collaboration, the future of DNA analysis promises even greater breakthroughs in solving crimes and uncovering vital information. “It's been fun to see that growth and be part of seeing that improvement. Hopefully, it'll continue to improve even more in the future, [with] lots of good people in the field, and I think that's going to see benefits for many years to come,” said Butler.

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