when TV shows like CSI: Crime Scene Investigation portrays DNA processing in the forensics lab, results appear instantly with a DNA match that closes the case within hours. However, the behind-the-scenes process of forensic DNA analysis is much more complicated, requiring careful interpretation of the data.

Forensic DNA profiling uses the polymerase chain reaction (PCR) to amplify short tandem repeats (STRs), which are highly variable regions of DNA, usually four repeating bases. This variability helps criminologist identify individuals. However, during PCR, the polymerase can slip over the repeats, creating “stuttering” peaks—fragments that are one repeat shorter than the true peak.

Since the advent of PCR, scientists have developed filters to handle predictable stuttering, but it remains a point of frustration while interpreting DNA profiles. “It’s especially hard when we’re dealing with mixtures of DNA,” he said Julie Sikorskya forensic scientist and manager of the Forensic Biology Unit of the Palm Beach County Sherriff’s Office. DNA levels can vary. “You have these trace contributors, and these allelic peaks can either be masked by stuttering, or you can interpret an allelic peak as stuttering.”

Many researchers have attempted to address this challenge, but have had limited success. Recently, at the International Symposium on Human Identification (ISHI), scientists at Promega Corporation announced a New DNA polymerase which drastically reduced stuttering.

“When we started our project a few years ago, we didn’t think it was possible to do it, because many people had tried this before and failed,” said Bob McLarenbiochemist at Promega.

When McLaren and his colleagues tackled the daunting task, they focused on a key question: Why does stuttering occur in the first place? “Our hypothesis is that strand slippage, or stuttering, is caused by the DNA sliding against the DNA polymerase it binds to, and that’s what’s doing the polymerization,” he said. David Mokrybiochemist at Promega.

They explored combinations of different polymerases and DNA-binding domains, focusing on structural motifs in nature. They were particularly attracted to the characteristics of T7 DNA polymerase – derived from the infecting T7 bacteriophage Escherichia coli-due to its ability to synthesize long stretches of DNA by binding and remaining attached for long periods.

The team focused on the thioredoxin-binding domain (TBD) of T7 DNA polymerase, which has a similar structure to that of most thermostable polymerases, including Taq. Thioredoxin, which is ubiquitous in cells, binds to TBD, triggering a conformational change that helps TBD enhance polymerase processivity. “So it was along the lines of thinking about whether we should improve processivity by having the DNA (polymerase) bind more tightly around the DNA in the active site,” McLaren said.

They set out to improve Taq polymerase by incorporating this feature from T7 DNA polymerase. They engineered a modified version of Taq polymerase by binding thioredoxin to the TBD and positioning the domain close to the active site, where the polymerase adds nucleotides to the growing DNA strand. Mokry set up their experiment, running the amplification with their chimeric DNA polymerase. Then he handed her his samples Nick Courtneya biochemist at Promega, to perform capillary electrophoresis, which separated the amplified fragments, and to examine the data peaks.

When Courtney saw the data, she couldn’t believe her eyes. “I took my laptop with me, ran to (David’s) office and was like, ‘Oh boy, you’ve got to see this,’ because not only did it work, it went from not working to work quite well. one day.” They achieved an approximately 85 percent reduction in stuttering across all loci. But the team wanted to improve it even more. Using machine learning, they analyzed the amino acid sequences to identify mutations that improved the binding of the polymerase to its template. They then tested their improved enzyme on mixtures with two and five contributors and observed a further reduction in stuttering to below-noise levels. Additionally, this enzyme works with a workflow and sensitivity comparable to traditional STR systems.

This development and refinement process took several years. When the team presented their much-anticipated breakthrough announcement at ISHI to the forensic science community, Sikorsky recalled the audience’s reaction as a mixture of stunned silence and excitement. After struggling with stuttering for decades, Sikorsky, who was not involved in the enzyme’s development, expressed optimism about the new enzyme and its potential to streamline DNA analysis for casework. “Being able to incorporate such a tool will revolutionize the field.” She added: “So not having to worry about this spike in stuttering increases confidence in our DNA interpretation, which allows us to have a more accurate number of contributor estimates.” Although the enzyme is not yet commercially available, the research team is working on creating a kit for forensic analysts to use.

Disclosure of conflicts of interest: Promega Corporation has applied for a patent for this enzyme for use in STR analysis.