Breakthroughs in Ancient DNA Analysis

How magnetic bead technology has facilitated the dawn of paleogenomics, unlocking secrets from ancient DNA fragments

By Kendra Atleework

It’s lonely at the top. Every once in a while, we Homo sapiens look around and remember that we are, in fact, the only extant member of the genus Homo. And then we get to wondering about the good old days, say 70,000 years ago when Homo sapiens made the acquaintance of Homo neanderthalensis, our closest known relatives. Researchers have long understood that portions of Neanderthal DNA persist in the modern human genome. And yet, without the ability to efficiently sequence ancient DNA, a deeper understanding of human evolution remained elusive.

In 2022, Svante Pääbo and his team shocked the scientific community and won a Nobel Prize® for a task thought to be impossible: the sequencing of the Neanderthal genome. When it comes to the study of ancient DNA, the secret to success lies in sample preparation. For his Nobel Prize winning work, Pääbo relied on target enrichment, PCR amplification, and massive parallelism via next generation sequencing using streptavidin-coated magnetic beads—breakthrough technologies now enabling researchers to work with more fragile or degraded DNA samples than ever before.

Pivotal techniques for the study of ancient DNA

In 1985, Svante Pääbo picked up what some might consider an unusual hobby: sequencing mitochondrial DNA from a mummy. He first experimented with mitochondrial DNA from an extinct animal that had waited behind museum glass for 140 years. Pääbo then progressed to working with nuclear DNA from Egyptian mummies. DNA from these early samples was heavily degraded and fragmented, with most sequences measuring around 100-200 base pairs (bp). Meanwhile, Typical DNA isolation yields fragments of 5,000 – 35,000 bp.

Such short sequences are par for the course when it comes to ancient starting samples. DNA fragments from samples between 4 and 13,000 years old regularly clock in between 40 and 500 bp, presenting a task beyond the scope of traditional Sanger sequencing. PCR primers, measuring around 20 bp in length, must bind to a specific matching sequence within a sample in order to amplify the DNA for purification and further experimentation. Ancient target DNA that has degraded to the same size—about 20 bp or shorter—presents significant obstacles.

The challenges of working with ancient DNA persist beyond amplification. Once amplified, DNA must still be pieced together into an entire genome. This is no small feat, given that one copy of the full Neanderthal genome rack up 3 billion base pairs. While Pääbo’s early work with ancient DNA was considered groundbreaking, newer techniques like target enrichment and next generation sequencing (NGS) were needed to propel his Nobel Prize-winning accomplishments.

Target enrichment

The process of target enrichment allows researchers to extract target molecules from a degraded and contaminated sample, in order to amplify target DNA. This process works a little like fishing. “You can bind bio-molecules to the surface of Dynabeads™ magnetic beads, like bait on a fishing line, in order to select exactly what you want to remove from a sample,” said Erlend Ragnhildstveit, R&D Director for Thermo Fisher Scientific, whose team develops Dynabeads magnetic beads products. “The bait, if you will, could be a certain antibody, a protein, it could be a DNA fragment.” Once baited, the beads are mixed with the sample. “Let’s say you have a sample of millions of different DNA pieces but you only want to isolate one particular type. Then you can use the specific DNA fragments that will only recognize and bind to that target of interest and pull that out of the solution using a magnet,” said Ragnhildstveit.

Pääbo’s team baited Dynabeads magnetic beads in such a way that they could select for mitochondrial DNA only from Neanderthals, and not DNA from contaminating bacterial DNA or other micro-organisms. Target enrichment via magnetic beads enabled Pääbo’s team to select only for the targeted DNA fragments.

Next generation sequencing and massive parallelism

When the pages of an ancient manuscript have crumbled, a narrative must be compiled using only fragments. So it is with ancient DNA. Before the technique of Next Generation Sequencing (NGS) was available, researchers relied on traditional methods to sequence DNA which would be too slow and expensive for an entire genome. “If ancient DNA is like a manuscript a researcher is trying to restore, you can only put together one phrase at a time using traditional sequencing methods,” said Ragnhildstveit. “Then you need to repeat that many times, with many machines in parallel, to restore the pages and eventually put together the entire book”.

To put this in perspective, 20 years ago using traditional methods, a lab would expect to spend six months and about $100,000,000 sequencing a human genome. Enter next generation sequencing, which allows short segments of sample DNA to be processed simultaneously with massive parallelism, thus efficiently piecing together much larger sections of genome sequences. “Next generation sequencing completely revolutionized this process using the concept of massive parallel sequencing,” said Ragnhildstveit. “If we’re thinking about ancient DNA as a manuscript, massive parallelism means you can read that entire book at the same time.” NGS utilizing Dynabeads magnetic beads has allowed researchers to dramatically improve throughput while dramatically bringing down costs.

Sample purity protection using KingFisher™ automation

While the spectre of sample contamination continues to haunt researchers, Dynabeads magnetic beads can be used in conjunction with other technology to protect ancient samples. The KingFisher Sample Purification System serves as a game-changer in the burgeoning field of paleogenomics by automating the DNA sample preparation process. “The Kingfisher purification system provides automation and reproducibility thus another level of contamination protection and workflow reliability for the sample,” Ragnhildstveit said. Used in conjunction with Dynabeads magnetic beads, Kingfisher automation helps scientists avoid wasting samples when working with precious ancient DNA.

A pinnacle in magnetic bead technology

Researchers working with ancient DNA have limited opportunities to extract information from rare and finite samples. Thus, the technology with which they approach the task is crucial. Streptavidin-coated beads are gentle on samples, as long as those beads are of a certain quality. Any craftsperson is only as good as their tools, and when it comes to the successful use of magnetic beads, consistency and reliability are key. “If your bead batch fails, that means a wasted sample,” said Ragnhildstveit. When it comes to working with ancient DNA, the stakes are especially high.

The processes of target enrichment and NGS are possible because of improvements in bead technology. Pääbo’s team used the product Dynabeads magnetic beads. “The beads were instrumental in enabling a technique where you could improve the isolation efficiency of longer strands of Neanderthal DNA, so you could get more information while being gentle on the sample,” said Ragnhildstveit.

Key features of magnetic beads:

Surface quality increases sensitivity

Improvements to surface chemistry of streptavidin-coated beads have enabled breakthroughs like Pääbo’s. Beads are a vehicle to move within a solution, and the sensitivity of a bead’s surface can predict a researcher’s success in isolating a target from a fragile and precious sample.

“The surface of the beads is the part that’s exposed to the sample,” Ragnhildstveit said. “The quality of that surface determines how effectively and specifically bio-molecules are going to bind.” Lower-quality bead surfaces can stick to unwanted material, such as contaminants in the sample, interfering with results.

“If the sample is a haystack, and the ancient DNA is a needle, it’s important that you pull out only the needle, and not the hay,” said Ragnhildstveit. “To prevent unwanted binding, the quality of the surface is very important.”

Consistency

The best streptavidin-coated beads, like the Dynabeads magnetic beads used by Pääbo’s team, are uniform in size and perfectly spherical, a consistency that offers researchers better batch-to-batch reproducibility. “Reproducibly is critical in all kinds of work, because you don’t want to introduce variables from the product itself,” said Ragnhildstveit. “You have to trust the product completely.”

Super-paramagnetism

Dynabeads magnetic beads are also super-paramagnetic, which means they are only magnetic when exposed to a magnetic field. Typically, a magnetized object remains magnetized, and some magnetic beads on the market retain their magnetism. But Dynabeads magnetic beads have the ability to lose that magnetism.

“Dynabeads magnetic beads lose absolutely all magnetism when you’re ready for that phase of the work,” said Ragnhildstveit. “That’s very important when you want to use them to isolate something like cells or DNA or proteins from a sample without also pulling down impurities due to clumping caused by remaining magnetism.”

What does this look like in the lab? First, magnetized Dynabeads magnetic beads are added to a sample, after being ‘baited’ to bind to a target. Once the beads bind, they are separated from the remaining sample solution and washed. At this point, they lose their magnetic charge, releasing from their clump and once again spreading apart.

Why do we still have Neanderthal DNA?

Breakthroughs in magnetic bead technology have allowed researchers to gain new information about ancient DNA. “It’s interesting to have the data,” Erlend says. “But it’s more interesting to understand what it means.”

Thanks to the burgeoning field of paleogenomics, we now know that about half of the Neanderthal genome still exists today, scattered amongst living human beings. Individuals of European and Asian descent each have about one-to-two percent of Neanderthal DNA, while people from parts of Oceania can trace up to five percent or so of their genetics to the Denisovans. Without a selective advantage, tens of thousands of years would have rinsed away most of Neanderthal and Denisovan DNA. Why, then, do these genes persist within the modern human genome?

Theories abound, and each one helps us to better understand ourselves. Perhaps, researchers posit, Neanderthal DNA conferred some advantages to modern humans when it came to surviving a colder climate. Denisovan genes may have imparted modern-day Tibetans with a proclivity for high-altitude survival.

As Svante Pääbo pointed out in his Nobel Prize lecture, the presence of ancient DNA in the modern human genome is also medically relevant to contemporary populations. Fascinatingly, each ancient gene that we carry forward represents a double-edged sword, conferring both protection and harm. In one example illustrated by Pääbo, a Neanderthal factor on Chromosome 3 leads to a risk of more severe covid, but also a decreased risk of HIV infection.

Additionally, in his Nobel lecture Pääbo pointed out that some facets of our reproductive health are also linked to our ancient relatives. A genetic progesterone receptor variant stemming from Neanderthals impacts fertility. This Neanderthal variant has been associated with pre-term births. Modern humans with this variant have a tendency to have premature babies—a seeming disadvantage. “You’d think that this gene would be lost during evolution because of selective disadvantage,” Ragnhildstveit said. But this variant also protects against miscarriage, resulting in more offspring and explaining its continued presence in our genome today.

“That finding was very important, because now we know that if a woman has the human variant of that gene, she’s going to be more at risk for miscarriage,” Ragnhildstveit said. “We know that if you treat a person with that variant with more progesterone, you can reduce that risk.”

Modern medical breakthroughs made possible by magnetic bead technology

By helping us understand our own genome, the study of ancient DNA can benefit modern healthcare outcomes. In addition, some technology used in the study of ancient DNA can have other lifesaving applications. For example, Dynabeads magnetic beads are being used in cell and gene therapy applications. “The use of magnetic beads has been revolutionizing a lot of areas,” said Ragnhildstveit. “Dynabeads magnetic beads are a dominant player in cancer diagnostics and testing.”

When it comes to early cancer diagnosis, Dynabeads magnetic beads also show promise in the realm of liquid biopsy. As cancer cells begin to grow in the body, each cell sheds a package of information into the bloodstream. With Dynabeads magnetic beads, the components of this information can be isolated and analyzed. When a liquid biopsy is performed, blood is drawn, and magnetic beads are used to check for the presence of cancer markers. This method may lead to earlier diagnosis than regular biopsy, revealing the presence of cancer markers before the cancer has developed into a more severe stage that is harder to combat. “This kind of biopsy is non-invasive and can be performed repeatedly,” Ragnhildstveit said. “Liquid biopsy using Dynabeads magnetic beads allows you to monitor cancer’s progression, and it also helps you track the efficacy of treatment.”

Paleontology and the source of human creativity

For over 400,000 years, Neanderthals existed on this earth. They ate, slept, and interacted with one another—much like humans. There’s even evidence that Neanderthals may have used language. But they differed significantly from modern humans in one crucial way.

They didn’t change. “They had the same tools at the beginning of their existence and at the end,” said Ragnhildstveit. “And that was the same all over the planet. Very little creativity, you could say. With modern humans, in 50,000 years, you see an enormous development.” Where Neanderthal art was simplistic, the art made by modern humans delved into the figurative where you can clearly see what is depicted. Where Neanderthals stayed in sight of land, modern humans crossed bodies of water. Where Neanderthal technology remained static, humans lost no time in whipping up something called the smart phone.

“Something changed in modern humans,” Ragnhildstveit said. Understanding that change is a driving force behind Pääbo’s work and the field of paleogenomics.

One hypothesis put forth by Pääbo’s team concerns genetic variants that determine neural development and cell division. “The hypothesis is that, because cell division takes longer, it’s a more precise, accurate process,” Ragnhildstveit said. “Perhaps if you have the modern human variant that determines cell division, you get fewer errors and a more functional result.” Perhaps you also get creativity, myth-making, and abstract thought, but that’s a question we’ll have to live with for the moment. In the meantime, even as tech advancements and bold researchers approach the bigger questions, we remain in many ways a mysterious to ourselves.

» Learn more about Dynabeads Streptavidin Magnetic Beads for Target Enrichment in the NGS workflow

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