Researchers have sequenced DNA fragments that date up to half a million years old, but these samples were obtained from the permafrost regions of Greenland, which preserved the genetic material from degradation like a gigantic laboratory freezer (1). Such preservation conditions are atypical when it comes to ancient DNA studies because most of the Earth has not been constantly frozen for millions of years. Furthermore, the genetic diversity present in these samples is limited, so researchers interested in specific mammals might have a hard time finding their samples in permafrost regions.
“You would have to find mammal bones in areas that would have been glaciated through the recent half million years,” says Matthias Meyer, an ancient DNA researcher at the Max Planck Institute for Evolutionary Anthropology, “which is highly unlikely because a mammal would just never go there.”
Permafrost aside, after years of improving the techniques for retrieval of genetic information from ancient samples, DNA can now be recovered and sequenced from samples up to 50,000 years old. As DNA molecules age after the death of an organism, they begin to degrade, becoming susceptible to strand breaks, miscoding lesions, and blocking lesions. While new recovery techniques and next-generation sequencing methods have overcome problems associated with strand breaks and miscoding lesions, blocking lesions—which prevent sequencing altogether—are still not fully understood or characterized.
Patricia Heyn, a former student assistant in Meyer’s lab, saw these blocking lesions as a potential target for new repair and recovery techniques. “We’re always trying to push the limits of ancient DNA research, to go back further in time and to analyze samples that are more degraded than the ones that we can currently work with,” says Meyer. To begin characterizing blocking lesions, Meyer and Heyn developed a polymerase extension profiling method to measure the frequency of these lesions in ancient samples (2).
Blocking the ‘Dark Matter’
Blocking lesions—DNA modifications that prevent polymerase bypass—prevent amplification, an essential step in next-generation sequencing, especially for ancient DNA samples which are usually of a limited quantity. Modified or lost thymines, oxidative base modifications, or inter-strand cross-links can act as blocking lesions.
Several previous studies estimated that a high percentage—about 90–95%—of all ancient DNA molecules have blocking lesions, making a majority of the genetic information inaccessible to even the most current high-throughput sequencing methodologies and PCR-based analyses. “That would have meant that there was a ‘dark matter’ of ancient DNA,” says Meyer, “DNA that we extract, that is present and preserved in the bone, but is inaccessible to DNA sequencing.”
Previous estimates on the abundance of blocking lesions in ancient DNA came from analytical chemistry techniques such as high-performance liquid chromatography and mass spectroscopy. But these approaches only provide an indirect measure of the frequency of block lesions. For example, one group fluorescently stained double-stranded DNA, denatured it by exposure to heat, and then measured the remaining fluorescent signal. “This is a very indirect approach, because there could be a lot of noise,” notes Meyer. Add to this the fact that analytical chemistry–based approaches cannot distinguish between DNA from the organism of interest and the other genetic material resulting from environmental contamination such as microbes that settle on the bone after death. Furthermore, the harsh buffers used in the sample preparation for these techniques could introduce additional, artificial damage.
Despite the need for a new way to access more genetic material from ancient samples, researchers had been unable to obtain a direct, high-resolution measurement of these blocking lesions while filtering out contamination. Sequencing could provide this measurement, but these block lesions prevent amplification and sequencing, rendering these degraded DNA strands invisible to researchers. “You could never see those blocking lesions because you lose the molecules,” explains Heyn. “It prevents amplification, and with a standard library preparation, you would never ever see those molecules.”
Sequencing the invisible
“We developed a special library that is way more complicated than a standard next-generation sequencing library to actually see those molecules in a direct way, with an end-of-template recognition sequence,” says Heyn. He and Meyer ligated these recognition sequences to the end of the DNA templates in ancient genetic samples. During amplification, the polymerase would be stopped from copying the recognition sequence on the strands with a blocking lesion. High-throughput sequencing would then be able to identify the strands without the recognition sequences, providing evidence of a blocking lesion in the sequence.
The protocol contains multiple purification steps, the development of which was no easy task, according to Heyn. To make sure these incomplete copies were not removed along with excess primers and extension products, the DNA strands were also ligated to biotinylated adapters that would bind to streptavidin beads during a later purification step. “We had to add an extra purification step because we figured out that there might be iron leaking from these magnetic beads and that this iron was poisoning the ligase,” says Heyn. “So, it took us a while to figure this out.”
The group worked on the project for two years, optimizing the protocol step-by-step for use with very low sample amounts. For Meyer, these extremely low amounts of starting material is one of the challenges of developing methods for ancient DNA studies. “We carefully thought of this protocol on paper, and when we tried to implement it in the lab, we were constantly challenged by new problems,” says Meyer.
After testing the protocol with templates with artificially created blocking lesions, the researchers aimed their polymerase extension primer technique at four ancient DNA samples: a 47,000-year-old ancient horse, a 27,000-year-old ancient horse, a 43,000-year-old mammoth, and a 42,000-year-old cave bear.
While these percentages are only based on the four ancient samples sequenced in this experiment, Meyer believes that these results will be typical of other ancient DNA samples. “This really came as a disappointment,” says Meyer. “If there is no more additional information preserved [in these samples], it’s basically a dead end.”
This could mean that the end is near for the development of significant ancient DNA recovery methods, according to Meyer. He has little hope that new methods will be able to recover ancient DNA from fossils that are much older than the ones currently accessible to genetic research. While other templates or repair enzymes could be applied to the primer extension method, it would only provide a better understanding of these blocking lesions, not a viable avenue to improve recovery.
The last unexplored avenue is nick repair, but Meyer is doubtful this will result in improved DNA recovery methods. While nicks can affect how many molecules are accessible to sequencing, nicks often occur in close proximity to one another, resulting in double stranded breaks. These small fragments of DNA can be handled by next generation sequencing. “I’m not very hopeful that nick repair would improve sequencing retrieval, but it’s something we haven’t actually investigated enough to come to a final conclusion.”
1. Lydolph M.C., J. Jacobsen, P. Arctander, M.T. Gilbert, D.A. Gilichinsky, A.J. Hansen, E. Willerslev, and L. Lange. 2005. Beringian paleoecology inferred from permafrost-preserved fungal DNA. Appl Environ Microbiol 71: 1012–1017.
2. Heyn, P., U. Stenzel, A.W. Briggs, M. Kircher, M. Hofreiter, and M. Meyer. 2010. Road blocks on paleogenomes—polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA. Nucleic Acids Research. 38: e161.
2010 marked the beginning of our Methods-specific Newsletter series. Covering cell culture, microscopy, PCR, and antibody technology, BioTechniques brought you the latest methodological and technical advances in these exciting fields through weekly feature articles and news stories. If you enjoyed the Top PCR Feature of 2010, check out the rest of the editors’ picks of our favorite methods-specific news features from 2010 here.