Modern man meets Neanderthal
Well, they actually did it. And the whole world knows about it now, even if the news is tens of thousands of years old. It was, of course, a juicy story for the world’s media: Neanderthals mated with modern humans! But for Svante Pääbo, Department Director at the Max Planck Institute for Evolutionary Anthropology in Leipzig, this is not the most important aspect of his discovery.
Bone can contain DNA from many organisms
Compared to the sequencing of the DNA of living individuals, a whole series of obstacles must be overcome before “old DNA” can be decoded. One of the main problems is that a sample from a 10,000-year-old Neanderthal bone may contain a lot of genetic material, but more than 95 percent of it does not belong to our caveman. “This is DNA from bacteria or fungi that have colonized the bone after death,” explains Pääbo.
The second problem is that, over time, the DNA strand disintegrates into smaller and smaller fragments, like a jigsaw puzzle whose pieces are scattered in the box. Some parts are still connected, but the overall picture (the sequence) is unrecognizable. The problem is compounded by the fact that some of the building blocks are chemically modified. Pääbo and his team discovered that one of the four bases in the genetic code changes its identity over time. Instead of a C for cytosine, many of the sequence fragments contain a U for uracil, especially at the ends of the fragments, where the cytosines lose their amino groups easily. During sequencing, the U is read as a T for thymine. “So when we find a T in the first position of a sequence, in 40 percent of cases, it is actually a C,” explains Pääbo.
A critical problem is caused by the researchers themselves: they can contaminate the samples with their own DNA. Individuals may leave small amounts of DNA behind, whether during the excavation at the archeological site or when preparing the gene fragment in the lab. This is helpful at the scene of a crime, but it can ruin the work of the paleogeneticists.
Pääbo and his team had to overcome all of these obstacles one by one. For example, the researchers succeeded in increasing the yield of Neanderthal DNA by a factor of 300. This was an important achievement as, before these advances, it looked as if the sequencing would take another two decades and cost 60 million euros.
The researchers enrich the Neanderthal DNA by using, for instance, molecular scissors that cut the microbial rather than the human genetic material. “In this way, we enrich the samples by up to 20 percent, which makes the sequencing affordable,” says Pääbo. To counteract the changed identity of the cytosine, the researchers wrote bioinformatics programs with which they corrected their computer algorithms for the sequencing to factor in the probability that a T was actually a C. “In a fragment that is 50 base pairs in length, this allows us to decide with confidence whether the DNA is from a Neanderthal or from a bacterium,” explains Pääbo.
Contamination by the researchers’ own DNA was a problem that the group had to grapple with for a long time. Numerous precautions were taken. This involves handling the samples in a clean room, similar to the type used in the chip industry. The air is constantly filtered and the scientists work in protective clothing. When the room is not in use, UV light destroys any remaining DNA. Yet all of these precautions were not sufficient for an experiment that the team carried out in 2006, details of which were published in Nature. “Given that we had taken these precautions, we thought that we had only 1 percent contamination. In fact, the figure was perhaps 14 percent,” says Pääbo. What happened?