Ancient DNA Extraction Laboratory
The Ancient DNA Extraction Laboratory (ADEL) is an interdepartmental, interdisciplinary Anthropology – Biological Sciences special use laboratory at Idaho State University. Principal investigator and research lead Dr. John Dudgeon performs original research and organizes and mentors student thesis projects using extraction, amplification and analysis of mitochondrial and genomic DNA products from old, degraded or fragmentary samples. The facility incorporates instrumentation, lab space and reagents obtained through startup monies, internal grants and in-kind loans and donations of equipment from other academic departments and campus facilities to establish new methods and techniques to extract molecular information from challenging archaeological remains, including human and animal bones and teeth, and stone and ceramic artifacts. At present, ADEL is not accepting contract work, but is interested in projects with high research visibility during the establishment of best practice analytical protocols.
The extraction lab is dedicated to ancient DNA extraction only. There is a class 100 positive pressure hood and one exhaust hood in the room. The extraction lab has a dedicated positive pressure air handling system to separate the extraction laboratory air supply from sample preparation (cleaning and LN grinding) and downstream PCR applications. The extraction lab is compact, with a footprint of only 97 square feet. While this makes it difficult for more than two persons to work in the lab at the same time, it permits fast and efficient cleanup and decontamination of work surfaces prior to extraction activities.
To prepare materials such as bone and tooth for DNA extraction, we use a SPEX 6770 freezer mill to grind samples into a fine, uniform powder which ensures that all apatite-bound DNA molecules can be accessed by an extraction buffer more rapidly than traditional, whole sample decalcification/extraction. Pre-freezing samples in sterilized polycarbonate sleeves prevents interaction between sample and liquid nitrogen (a known source of exogenous DNA contamination), and permits faster grinding without generating DNA-damaging heat. Bone and tooth powders are ground to 50-100 µm, optimal size classes for efficient decalcification.
To purify extracted DNA we use the Promega Wizard® DNA Clean-Up System which uses a Vac-Man® vacuum manifold to draw samples through silica membrane minicolumns to bind DNA, allowing us to concentrate, remove contaminants and desalt the final product. In consultation with (Dr. Brian Kemp (Washington State University), we are working on methods to reduce the total extraction to very small volumes (< 2 ml), which improves DNA yields and eliminates unnecessary extraction transfer or volume reduction steps.
The PCR lab is physically separate from the grinding/prep lab and extraction lab and has two class 100 positive pressure hoods and one carbon filter recirculating exhaust hood. One positive pressure hood is dedicated for setting up PCR reactions, the other houses an MJ Research PTC-200 Peltier thermal cycler with heated lid (on long term loan from Idaho State University's Molecular Research Core Facility (MRCF).
Short-term sample storage is accommodated in a large, 37 cubic foot 4 C refrigerator, and longer-term storage in a benchtop -20 C freezer. A current pressing need is a chest-type -80 C freezer for long term storage. Future grant applications will address this deficiency; however, -80 C long-term storage is available in departmental common use facilities in Biological Sciences.
We also have a gel electrophoresis station with a mini gel rig, EtBr staining area and a homemade UV light table and camera documentation system that we use to vizualize PCR products. We are very proud (and a little surprised) by the excellent images we generate from our gels (below). Other researchers have remarked on the superior band visualization we achieve with our cobbled together system, often eclipsing the results from their purpose-built UV visualization equipment. Below are images of a 142 bp fragment from several samples of c. 500 ybp Rapa Nui (Easter Island) mtDNA (00008F – 00151R, HVR-II; Left), and the results of microsatellite fragment PCR of the same samples using Applied Biosystem's Minifiler® reduced primer length multiplex (Right).
As mentioned above, we are currently working on several projects to recover and amplify DNA from challenging archaeological and forensic samples from Dr. Dudgeon's original research, as well as projects involving colleagues from ISU, Ohio State University and International Archaeological Research Institute, Inc. Some of our current project load:
- Mitochondrial and microsatellite ancient DNA analysis of Rapa Nui (Easter Island) kinship in the late prehistoric and early protohistoric periods (c. A.D. 1550 - 1860)
- Mitochondrial ancient DNA analysis of prehistoric Fijian hillfort skeletons (c. A.D. 1250 - 1600)
- Mitochondrial ancient DNA recovery and analysis from stone projectile points from the El Mirador site, Petén, Guatemala, a putative Late Classic (A.D. 600 – 900) Maya battlefield; we will attempt to discern whether diagnostically Maya projectile points contain Mexican (central highland Mexico) blood residues, and vice versa
- Mitochondrial DNA analysis of chicken and pig bones from Hawaiian archaeological sites (c. A.D. 1500) to investigate prehistoric evidence for ceremonial exchange within Hawaii Island (Big Island)
- DNA extraction and human loci testing of purported human “shrunken heads” from the Central American Ethnographic collections, Idaho Museum of Natural History
- Mitochondrial DNA analysis of small mammal bones and teeth from raptor caves in the eastern Snake River Plain; we will examine demographic changes in genetic diversity correlated with stable isotope indicators of climatic shifts in the early and middle Holocene
We are deeply grateful for the efforts of ISU's Molecular Research Core Facility, who perform our sequencing and fragment analysis using an Applied Biosystems 3130XL Genetic Analyzer. The MRCF at ISU is smaller than many larger university molecular core facilities, but is very responsive, conscientious and accommodating of our ancient samples, allowing us to perform on-the-fly extraction and PCR optimization to work through challenging samples. Examples of fragment analysis (top) and sequencing (bottom) data from the core facility's capillary electrophoresis instrument demonstrates the results of our close partnership with the MRCF for our ancient Rapa Nui samples.
Three publications are nearing completion/journal submission from ADEL, as well as several upcoming conference presentations that demonstrate the versatility of our methodological improvements to the extraction and amplification of DNA from old, degraded and fragmentary samples. Check back soon for updates and pdf reprints of this research.