Developing Tools for Extraction and Sequencing of DNA from Fossil Samples

In recent years, the popularity of investigating biomolecules such as proteins in soft tissues from fossil samples has grown significantly. However, research on other macromolecules, like nucleic acids, found in fossil samples is relatively scarce. Schweitzer and colleagues have demonstrated the ability to stain for DNA in fossil dinosaur bones (Schweitzer 2013, Bailleul 2020), which has been repeated by other groups . Yet, published reports of sequencing attempts are scarce, which may result from assumptions about the age of fossils within the conventional old earth paradigm and whether intact DNA can be extracted from fossilized material. However, in a young earth model, it is possible that DNA could survive in fossilized materials in trace amounts and be studied. Further, any method that is used to analyze this DNA would need to reduce contamination or be able to distinguish between contaminant and original source DNA. A novel, benchtop, method of DNA sequencing has been developed in recent years: Oxford Nanopore Sequencing (ONS). This method avoids the replication of damaged or short DNA strands as it does not require PCR. This also potentially prevents the oversampling of contaminant DNA (eDNA, human contamination, etc.) by avoiding a PCR amplification step. Furthermore, ONS is also relatively inexpensive compared to traditional techniques. To test ONS on ancient samples, several fossil samples of varying ages from 500 years old and beyond were obtained through collaboration. Samples were primarily prepared through physically crushing or through core drilling to expose internal portions of the fossil. Then, after the soaking of the sample in buffer and post-soak filtering, the filtrates were examined using a double-stranded DNA (dsDNA)-specific fluorescent dye. Two fossil samples have been tested so far including an ~500-year old cow femur and a ~2,000 year old pig rib bone both from England. Both samples have already been shown to have endogenous collagen present. Further, both samples were found to have dsDNA in samples that were prepared through grinding of the bone. DNA sequencing of these samples demonstrated both samples yielded DNA fragments (cow femur mean fragment length = 80-110 bp; pig rib mean fragment length = 100-200 bp). Metagenomic analysis provides evidence of some contaminant DNA fragments of bacterial, human, or plant origin. However, most of the sequence fragments were not identified. While analyses are preliminary, the evidence indicates that the extraction and sequencing of intact DNA from fossil bones is possible. In addition, this work provides the foundation for testing fossils of increasing age using additional protocol refinements with the intention of sequencing DNA from fossils formed during the Flood and the post-Flood Ice Age including dinosaurs. References: Schweitzer, M.H.; Zheng, W.; Cleland, T.P.; Bern, M. (2013) Molecular analyses of dinosaur osteocytes support the presence of endogenous molecules. Bone 52, 414-423, doi:https://doi.org/10.1016/j.bone.2012.10.010. Bailleul, A.M.; Zheng, W.; Horner, J.R.; Hall, B.K.; Holliday, C.M.; Schweitzer, M.H. (2020) Evidence of proteins, chromosomes and chemical markers of DNA in exceptionally preserved dinosaur cartilage. National Science Review 7, 815-822, doi:10.1093/nsr/nwz206.