School of Human Evolution and Social Change, Arizona State University
Friday All day, Clinch Concourse
The ability to detect ancient DNA (aDNA) in archaeological samples is a crucial step in the process towards implementing enrichment and sequencing strategies. Quantitative real-time PCR is a commonly used tool in aDNA studies used to detect, authenticate, assess quality and quantify ancient template. But there are significant challenges to creating reliable assays, choosing appropriate user-based parameters and interpreting the output for low quality samples.
Here we report the results of two case studies. We tested over 100 human skeletal remains displaying lesions characteristic of miliary tuberculosis with three qPCR assays (IS6110, IS1081 and rpob) targeting single and multi-copy regions within the MTBC. The obstacles for detecting ancient pathogen DNA are exacerbated by the existence of a wide range of unrelated environmental Mycobacteria, making an assessment of MTBC presence, sequence specificity, and quality a non-trivial matter. We also tested the skeletal remains of four chimpanzees from Gombe National Park, Tanzania, with a qPCR assay targeting a small region of the Pan troglodytes mtDNA genome. Our assay indicated that none of the chimpanzee samples was positive for the endogenous target.
While it is claimed that double stranded DNA-binding dye (e.g. SYBR Green) is a more cost-effective and sensitive qPCR method, our results support the use of fluorescent reporter probes (here, Taqman technology) for acquiring highly specific PCR product in low quantity template, and for effectively assessing sample preservation. Downstream enrichment procedures and sequencing validate the results yielded by our qPCR assays, helping to authenticate any aDNA determination.
This work was supported in part by grants from the Leakey Foundation and the National Science Foundation to ACS, and National Science Foundation Graduate Research Fellowship grants to KMH and MANC.