Blake C. Ballif, Microbiology
Introduction
During the past decade, the development of ancient DNA research has left modern science seemingly on the verge of breaching the barrier between the world we live in and the ancient past. Archaeologists, anthropologists, and molecular biologists alike have become increasingly more aware of the understanding that can be gleaned from genetic studies of ancient populations and extinct species. Although ancient DNA has remarkably been isolated and amplified from organisms hundreds, thousands, and even millions of years old 1·3, these breakthroughs represent in most cases a single ancient individual or animal with important yet modest amounts of data. In order to perform the more informative population- sized studies of ancient peoples or species, sufficient quantities of DNA must be isolated from larger numbers of individuals in a reproducible manner.
Originally, the specific goal of my research was to use DNA analysis to perform ancient “geneology,” by reconstructing the genealogical relationships that might exist within a group burial of fifteen Egyptian mummies excavated in 1994 from the Fag el Gamous cemetery in the Egyptian Fayum by a team of BYU researchers that included Dr. Scott R. Woodward and myself. Of particular interest to this study is the fact that two children were burled at either side of one of the adult burials. Some of the questions we hope to address through DNA analysis are: Does this represent a father or mother and his or her children? If not, are the two children related? Does this entire group burial represent a family plot? Do the individuals buried in shafts in the sand contain succeeding generations?
As intriguing as these questions might be, the degraded nature of ancient DNA often causes only rare amplifications and results in the loss of irreplaceable tissue samples during unsuccessful DNA extractions. The relatively low success rate for extraction and amplification of ancient DNA experienced by all ancient DNA researchers Jed us to pursue improvements and alternatives to our current procedures. With the help from funds provided by the BYU Office of Research and Creative Activities, improvements in extracting and amplifying ancient DNA from 2,000 year-old Egyptian bone and teeth samples have been made.
Repair PCR
It has been theorized by some experts that, under normal conditions, DNA will last no longer than 50,000 years before becoming completely degraded’. However, the extremely rare conditions under which certain specimens have been preserved, along with the fact that DNA has been successfully extracted and amplified from such ancient samples, indicate that DNA can remain intact much longer. Yet it is evident that a great deal of damage has occurred to the DNA resulting in the recovery of DNA fragments only a few hundred base-pairs in length5. DNA fragments may also contain numerous lesions where individual nucleotldes have been damaged or lost. These missing bases could cause the DNA to break down into even smaller fragments during the denaturing step of PCR. In order to remedy this situation, it was hypothesized that subjecting the ancient DNA extract to a “repair” PCR reaction prior to the formal amplification process would allow the DNA polymerase to fill in any gaps or holes left by damaged or missing nucleotides. The “repaired” DNA could then be more successfully amplified by normal PCR.
To study the effects of repair PCR. DNA was extracted from the teeth, bone, or tissue of ten different ancient Egyptians found in the group burial mentioned previously using a standard technique involving guanidine isothiocyanate as a protein denaturer followed by the adhesion of DNA to glass beads’. Forty cycles of normal PCR resulted in no amplification from any of the samples. The same samples were then subjected to repair PCR which involved the removal of PCR primers to prevent amplification while the Taq polymerase was allowed to replace missing nucleotides. Primers were then added, and the PCR reaction was continued for forty more cycles. This reaction resulted in the amplification of DNA from three of the ten samples.
One of the amplified segments of ancient DNA came from the adult (identified as SE I) mentioned above and the second from one of the two children buried at its side (identified as SE3). The third amplified sample represents the DNA from another adult (identified as SE II) which was buried only a few feet away from the adult and child. The amplified fragment of DNA represents a segment of the mitochondrial D-loop region that is a noncoding region and has a very low selective pressure on mutations. Since mitochondrial DNA is strictly maternally inherited it is important in determining familial relationships and can be used to identify individuals with the same recent maternal lineage 7
DNA sequence analysis of the three segments amplified by the repair PCR reaction indicated that none of the three ancient Egyptians shared a common maternal ancestor (See Table 1). However, until DNA sequence is obtained from the other child we can not rule out the possibility of the two children being related. The physical remains of the adult (bone structure and the presence of a beard) indicate that it was a male, and since mitochondrial DNA is only passed on from mother to child, we still can not rule out the fact that the adult could be the father. In which case, another region of DNA must be amplified, such as the nuclear HLA locus which would reveal the genetic contribution of both parents. All things considered, the fact that the repair PCR technique increased amplification of ancient DNA by 30% is extremely important. This relatively simple procedure shows that repair of ancient DNA is possible and that future experiments could lead to the development of even more efficient ancient DNA repair protocols.
Microfiltration and “Magic Preps”
Athough in the past scientists have been successful in extracting ancient DNA from archaeological remains, new techniques with potentially higher success rates are always welcome. In an effort to increase the amount of ancient DNA recovered during the extraction process, two techniques involving rnicrofiltration and “Magic Preps” (manufactured by Prom ega) were adapted from protocols described by Hardy et al’ and N. Tuross6 respectively. Microfiltration operates on the idea that solutions used to extract the DNA from bone can be passed through a microporous filter which traps DNA fragments greater than fifty base-pairs in length. The DNA can then be washed off the filter and collected for PCR.
“Magic Preps” consist of synthetic resin beads that bind small fragments of DNA with high efficiency. When mixed with a solution that has been used in extracting DNA from bone, any DNA in the solution will become bound to the resin. The resin can then be collected in a special column to which it specifically binds, and the DNA can then be eluted off into sterile water for use in PCR. Since the majority of archaeological samples consist of ancient bone, and since some ancient bone has proven to be less effective in preserving ancient DNA than other tissue types (such as teeth10), it was anticipated that the two techniques described briefly above would be more successful in yielding amplifiable DNA from ancient bone fragments.
To test the productivity of the two new techniques, five bone samples representing five random ancient Egyptian individuals were crushed with a sterile mortar and pestle until approximately one gram of bone powder was obtained. The bone powder was collected in a 15 ml tube and decalcified at 37′ C in a total of 20 ml of 0.5 M EDTA pH 8.5 over a period of 2-3 days or until the EDTA wash was clear of colored matter. The EDTA washes were collected in 50 ml tubes and stored at 4′ C. Following these washes, 2.5 ml of a proteinase K solution was added to the remaining bone powder and incubated at 37′ C overnight. The supernatant was then phenol: chloroform extracted twice and the aqueous phase containing the extracted DNA was trapped by microfiltration using Centricon-30 devices manufactured by Amicon. The DNA extract was then washed off the filter in sterile water. Of the five samples subjected to this method of extraction, two eventually produced PCR amplified DNA fragments, one of which required the use of the repair PCR method previously described.
The EDTA washes collected during the decalcification step described above, were believed to contain some ancient DNA as well. Therefore, three of the five EDTA washes were prepared for DNA extraction with “Magic Preps” by concentrating the large volume of EDTA wash from 20 ml down to less than 1 ml using Centriprep-30 or Centriplus-30 microconcentrators. These samples were then treated with “Magic Preps” and the extracted DNA was eluted from the resin in sterile water. Treatment with “Magic Preps” resulted in one more PCR amplified DNA fragment.
Another procedure that was found to be useful after the extraction of DNA with either of the above methods was the visualization of the amount of DNA in the extract by running 20fLl of the extract on a 2.5% Nusieve/ME Agarose gel and staining with ethidium bromide. The amount of DNA in the extract could then be estimated by how bright the extract fluoresced orange under ultraviolet light. Inhibitors to the PCR, that often copurify with ancient DNA extracts, can also be visualized in this manner since they fluoresce blue under ultraviolet light. This simple technique helped in determining how to properly approach the PCR reaction for optimal results (i.e. diluting out the inhibitor, increasing the amount of DNA added to the PCR reaction, or by performing repair PCR).
Even though the improvements were not as drastically better as we would have hoped, the procedures were successful and a great deal of information about the quality and quantity of DNA that can be extracted from ancient bone has been gained in the process.
Conclusion
Athough the potential to more fully understand our past through the emerging field of ancient DNA research is exciting, reconstructing genealogical relationships through ancient “gene-alogy” is only one of the many intriguing applications of this new and powerful technology. Up until now, the study of ancient DNA by the scientific community has mainly been limited to isolated archaeological remains and museum specimens. The numerous mummies at the Fag el Gamous cemetery which is currently being excavated by BYU offer a unique and pristine population tied to many of the interesting historical events of ancient Egypt. Support of this project by the BYU Office of Research and Creative Activities has helped in providing valuable progress toward more effective methods for ancient DNA analysis which will eventually and undoubtedly lead to Invaluable Insights into what life was like for many of our ancient ancestors.
Acknowledgements
I would like to recognize Joel Myres who also assisted with the DNA extractions and sequencing.
Notes
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