Ancient DNA (aDNA) is DNA taken from very old sources, such as remains or environmental DNA. Because of breaking down over time, ancient DNA is more damaged than DNA from today. Scientists have found genetic material in old bones, mummies, old medical samples, plants, ice, permafrost, lake and ocean sediments, and dirt from excavations.

Even with the best protection, DNA older than 0.4 to 1.5 million years is hard to study. The oldest DNA found in physical samples is from mammoth teeth in Siberia, over 1 million years old. In 2022, scientists found DNA that is 2 million years old in Greenland sediments. This is the oldest DNA discovered so far.

History of ancient DNA studies

In 1984, the first study of what would later be called ancient DNA (aDNA) was conducted by Russ Higuchi and colleagues at the University of California, Berkeley. They found that DNA from a museum specimen of the Quagga, a type of zebra that is now extinct, remained intact more than 150 years after the animal died. They also successfully extracted and sequenced the DNA. Over the next two years, Svante Pääbo studied naturally and artificially mummified specimens and confirmed that DNA could be preserved in human remains that were thousands of years old, not just recent museum samples.

At the time, sequencing aDNA required difficult processes involving bacterial cloning, which slowed progress in the study of aDNA and the field of museomics. However, the development of the polymerase chain reaction (PCR) in the late 1980s allowed scientists to study aDNA more efficiently. Techniques like double primer PCR amplification (jumping-PCR) were used, but they sometimes created incorrect DNA sequences. To solve this, scientists used a method called multiple primer, nested PCR.

After the introduction of PCR, many research groups published findings about isolating aDNA from very old specimens. Some studies claimed to extract DNA from organisms that were millions of years old, including insects, plants, and bacteria preserved in amber from the Oligocene and Cretaceous epochs. Similar claims were made about DNA from dinosaur bones and eggs, as well as from halobacterial sequences in halite dating back 250 million years.

As scientists learned more about how DNA is preserved and the risks of contamination, they began to doubt the validity of these claims. Many studies could not be replicated, and most of the claims about aDNA older than a few million years were later dismissed as incorrect.

In 2007, a new method called single primer extension amplification was introduced to address damage to DNA caused by aging. Since 2009, advances in cheaper research techniques allowed scientists to study aDNA more effectively. By 2010, researchers could sequence complete genomes from ancient human remains. High-throughput Next Generation Sequencing (NGS) became essential for reconstructing the genomes of ancient or extinct organisms. A method for preparing single-stranded DNA (ssDNA) libraries also gained attention among aDNA researchers.

In addition to these technical improvements, scientists began to develop better standards for evaluating DNA results and understanding the challenges involved in aDNA research.

In 2022, Svante Pääbo was awarded the Nobel Prize in Physiology or Medicine for his discoveries about the genomes of extinct hominins and human evolution. A few days later, a study in Nature reported the discovery of two-million-year-old genetic material in Greenland, which is currently considered the oldest DNA found to date.

Problems and errors

Ancient DNA is of lower quality than modern DNA because of breakdown processes such as chemical changes that cause DNA to break apart. These changes and how well ancient DNA can last over time limit the types of scientific studies that can be done and set a maximum age for DNA samples that can be successfully analyzed. While there is a general connection between how old DNA is and how much it has broken down, differences in the environment where the DNA was stored make this relationship harder to predict. DNA samples that were stored in different conditions may not follow the same pattern of aging and breakdown. Even after being found, DNA can break down faster if stored in conditions that change, such as varying temperatures or humidity. Even with the best preservation, DNA from samples older than 0.4 to 1.5 million years may not have enough material for modern sequencing methods.

Studies on the breakdown of DNA in moa bones have shown that mitochondrial DNA, a type of DNA found in cells' energy-producing parts, can break down to an average length of 1 base pair after 6,830,000 years when stored at −5 °C. These findings were made by experiments that speed up the aging process, showing that storage temperature and humidity strongly affect DNA breakdown. Nuclear DNA, which is found in the cell's nucleus, breaks down at least twice as fast as mitochondrial DNA. Earlier claims of finding DNA from much older remains, such as Cretaceous dinosaur bones, were likely due to contamination from other sources.

A review of research on ancient DNA shows that few studies have successfully recovered DNA from remains older than several hundred thousand years. Greater awareness of the risks of contamination and studies on DNA’s chemical stability have raised doubts about some earlier findings. For example, DNA once thought to come from dinosaurs was later found to be from humans. DNA found in halobacteria that was enclosed in protective layers has been criticized because it is similar to modern bacteria, suggesting possible contamination, or it may have come from slow, long-term activity by the bacteria.

Ancient DNA can contain many errors that occur after death, and these errors increase over time. Some parts of DNA are more likely to break down, leading to incorrect data that may not be caught by tools used to verify results. Because of errors during sequencing, scientists must be very careful when interpreting data about population sizes. Errors where the letter "C" in DNA changes to "T" and "G" changes to "A" are very common in ancient DNA samples.

Another challenge with ancient DNA is contamination from modern human DNA or from microbes (many of which are also ancient). Recent methods have been developed to reduce contamination, such as extracting DNA in very clean environments, using special tools to identify DNA that originally belonged to the sample, and using computer analysis to compare results with known DNA sequences to estimate how much contamination may have occurred.

Authentication of aDNA

In the 2000s, progress in ancient DNA (aDNA) research made it more important to verify that recovered DNA is truly ancient and not contaminated by modern DNA. Over time, DNA breaks down, and the building blocks called nucleotides can change, especially at the ends of DNA molecules. One key change is when cytosine (C) turns into uracil (U) at the ends of DNA. During DNA sequencing, enzymes called DNA polymerases pair adenine (A) with uracil (U), which creates a change in the aDNA data, turning cytosine (C) into thymine (T). This change happens more often in older samples. Scientists can measure how often C turns into T using tools like mapDamage2.0, PMDtools, or metaDMG. Another process called hydrolytic depurination causes DNA to break into smaller pieces, creating single-stranded breaks. These small fragment sizes, combined with the pattern of chemical changes, help scientists tell ancient DNA apart from modern DNA.

Non-human aDNA

Although there are challenges with ancient DNA, more and more aDNA sequences are being published from various animals and plants. Scientists study different materials like mummified remains, bones, shells, ancient feces, alcohol-preserved specimens, rodent middens, dried plants, and recently, DNA from soil samples.

In June 2013, researchers including Eske Willerslev, Marcus Thomas Pius Gilbert, and Orlando Ludovic from the Centre for Geogenetics, Natural History Museum of Denmark at the University of Copenhagen, announced they had sequenced the DNA of a 560,000 to 780,000-year-old horse. The DNA was extracted from a leg bone found in permafrost in Canada's Yukon territory. A German team also reported in 2013 the reconstructed mitochondrial genome of a bear, Ursus deningeri, more than 300,000 years old. This showed that ancient DNA can be preserved for hundreds of thousands of years outside permafrost. In 2021, scientists reported nuclear DNA from the teeth of two Siberian mammoths, both over a million years old, preserved in permafrost.

In 2016, researchers measured chloroplast DNA in marine sediment cores and found diatom DNA dating back 1.4 million years. This DNA had a half-life significantly longer than previous research, up to 15,000 years. Kirkpatrick's team also found that DNA decayed at a half-life rate until about 100,000 years, after which it followed a slower, power-law decay rate.

Human aDNA

Because many people are interested in human remains, scientists have studied them closely to learn about DNA. Contamination is a big problem because the researchers and the remains come from the same species.

Mummies have been used for DNA studies since the 1990s and 2000s because their bodies are well-preserved. Examples include Ötzi the Iceman, who was frozen in a glacier, and bodies from the Andes that dried quickly at high altitudes. Other preserved tissues, like those from ancient Egyptian mummies, have also been studied. However, mummies are rare. Most DNA studies use bones and teeth instead, which are more common in archaeological finds. The petrous bone in the ear is often used because it is dense and holds DNA well. Other sources, like ancient feces and hair, have also been used. Contamination remains a major challenge in these studies.

Ancient DNA from diseases has been found in samples over 5,000 years old in humans and up to 17,000 years old in other species. Scientists have studied DNA from mummies, bones, teeth, and other tissues like calcified pleura, paraffin-embedded tissue, and formalin-fixed tissue. Tools like QIIME and FALCON help analyze this DNA efficiently.

In 2012, scientists studied Neanderthal bones from El Sidrón cave and found new information about their family relationships and genetic differences. In 2015, researchers discovered DNA from a Denisovan hominin in a 110,000-year-old tooth.

These studies have changed how scientists understand how humans populated Eurasia. A 2018 study showed that a large migration during the Bronze Age greatly changed the genetic makeup of the British Isles, bringing the Bell Beaker culture from Europe.

Ancient DNA has also shown connections between Central Asian ancestors and the first people in the Americas. In Africa, DNA breaks down quickly in warm climates, but scientists found 8,100-year-old DNA samples in 2017.

Ancient DNA has helped scientists estimate when modern humans started to separate. By studying DNA from Stone Age hunter-gatherers and Iron Age farmers, researchers found that human groups began to split between 350,000 and 260,000 years ago.

As of 2021, the oldest complete human genomes are about 45,000 years old. This information helps scientists understand human migration and history, including how early humans mixed with Neanderthals.