University of Pennsylvania Museum of Archaeology and Anthropology

Colonial DNA at Our Finger- (and Toe-) Tips – By Raquel Fleskes


By: Anne Tiballi

August 16, 2016

In the middle of the sticky mid-June heat, I arrived in Knoxville, Tennessee, with six skeletal samples, a car full of chemical reagents, and the mindset to learn how to do ancient DNA extraction. With help from the Penn Museum, I was able to travel to the University of Tennessee at Knoxville to work with Dr. Graciela Cabana and her graduate student, Frankie West, to learn how to extract DNA from skeletal material.

University of Tennessee (Photo by R. Fleskes)
University of Tennessee. Photo by Raquel Fleskes.

I brought with me six skeletal samples from a 17th-century historic site called Avery’s Rest, in Sussex County, Delaware, currently housed at the Smithsonian’s National Museum of Natural History in Washington, DC. The Avery’s Rest archaeological site represents an early English settlement in what is now the Rehoboth Beach area. The site was first occupied in 1674 by John Avery and his family, and remained an active tobacco plantation until 1715, when the land was sold  by John Avery’s surviving daughters, 27 years after his death.

 

The site provides a unique opportunity to explore labor and kinship relations on a 17th-century English plantation during an era of immense population movement and settlement in the Atlantic world. My aim was to see how mitochondrial DNA could be used to study these themes by providing information on maternal genetic relatedness between the individuals buried at the site, and find out where these trans-Atlantic colonists came from using genetic ancestry. These questions have traditionally been answered using historical documents and archaeological information, but new evidence generated through genetics has the capacity to reveal new interpretations.

Avery’s Rest bone samples before DNA sampling. Photo by Raquel Fleskes.
Avery’s Rest bone samples before DNA sampling. Photo by Raquel Fleskes.

To answer these questions, I brought metatarsal or metacarpal samples from six of the buried individuals at the Avery’s Rest site to the ancient DNA (aDNA) lab at UT. The aDNA lab is a small room, divided into smaller rooms, designed to prevent and reduce any foreign contamination by modern DNA. Before going into the ancient DNA lab, I had to cover my entire body with PPE (or personal protective equipment), putting on a full white hazmat suit, shoe covers, a surgical mask, a face shield, and two pairs of gloves. Needless to say, I looked like a giant white marshmallow most of the time!

The first step was to cut and weigh out the bone samples. I did this using a dremel saw, slicing the bone into small segments. To ensure there was no cross-contamination between samples, all equipment and the space was cleaned between each sampling. The bone was then soaked in bleach to  remove all surface contamination it might have been exposed to after excavation. Then the bone was thoroughly washed with water to remove any bleach residue and set to dry.

After the bone was bleached, it was ready to be powdered. This is done using a Freezer Mill, which chills the bone fragments using liquid nitrogen to make them easier to pulverize via a metal pellet that oscillates back and forth very quickly to grind the bone down. Pulverizing maximizes the DNA extraction process by increasing the exposed surface area of the bone.

Samples before they are inserted into the Freezer Mill. Freezer Mill pictured in back, with liquid nitrogen being poured into the machine. Photo by Raquel Fleskes.
Samples before they are inserted into the Freezer Mill. Freezer Mill pictured in back, with liquid nitrogen being poured into the machine. Photo by Raquel Fleskes.

Following the lengthy sample prep procedure, the bone was ready for DNA extraction. We followed the protocol outlined by Dabney et al. 2013 [1], which involves first incubating the bone powder at 56C degrees for 24 hours in an EDTA and Proteinase K extraction buffer. This pulls the DNA out of the bone powder and puts into a solution so it can be isolated. The solution is then combined with a binding buffer, which binds to the DNA. This, in turn, is spun down rapidly using a spin-column, which contains a small filter in it that catches all the DNA as the solution runs through it.

The filter that contains the DNA is then washed multiple times to remove any remaining solution residue, and then dried – this is called the DNA purification step. By the end of this process,  theoretically only the DNA will remain in the filter. Then the DNA is eluted – or removed – by running another buffer through the filter. Voila! The DNA has been extracted.

This process took me a whopping five days to complete. But it was not the end of the ancient DNA experience – once you had the DNA extract from the samples, the DNA has to be amplified and then sequenced, which took another four days. For this project, I wanted to sequence the mitochondrial DNA (mtDNA) control region – mtDNA is copied directly from each individuals mother, so it is useful for understanding mutational sequences in maternal ancestry. To accomplish this, I used 11 primer pair sets which targeted the mtDNA control region and amplified them using PCR.

The DNA amplifications went beautifully – the bone samples were very well preserved and I was able to extract and amplify large amounts of DNA from them without any external contamination!

The work went so well that I am planning on returning in a couple of weeks to extract DNA from the remaining individuals at the site.  It has been a whirlwind of an experience learning how to extract DNA from skeletal bone, and has rewarded me with exceptionally preserved DNA. I am very thankful for all the training and advice by Frankie and Dr. Cabana at UT, and the financial support of the Penn Museum to be able to pursue this research.

References Cited:

[1] Dabney, J., Knapp, M., Glocke, I., Gansauge, M.-T., Weihmann, A., Nickel, B., … Meyer, M. (2013). Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proceedings of the National Academy of Sciences of the United States of America, 110(39), 15758–63.


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