Christi Embry and Dr. Ray Matheny, Anthropology
Molecular biological techniques are allowing researchers to answer questions hidden within the physical archaeological record. DNA analysis of individuals excavated from multiple ancient populations has previously shed light on the origins and genetic affinities of these ancient peoples (5,6). These studies have the power to determine the structure of the ancient populations and may identify modern populations that have descended from them. Laboratory techniques have allowed researchers to identify four major haplogroups (A, B, C, D) and one odd group called X, that make up the majority of the modern Native American populations in North and South America. (8) This same haplotype analysis of ancient samples will yield insight into the relationship between the ancient and modern populations.
I proposed that we apply DNA analysis techniques to individuals found at the Tucume site in Peru. Peruvian archaeologists have been excavating the Pyramid complex of Tucume since 1988 (4). They have dated occupation of the complex from pre-Inka to Inka times (AD 1405). Within the walls of the most elaborate building were the remains of 22 tejadoras, or weavers’ (4). Although unsure about the exact original ethnic affiliation of these women, it is thought that the possession of these weavers was a sign of power. Archaeological evidence suggests that they were the royal weavers of the local leader. When he died they were killed and buried with him (4). The question of where they came from remains unanswered. How are they related if at all? It is hypothesized that they came from the surrounding villages. If this is the case then they may have been given to the leader as a symbol of loyalty, or they may have been taken from their native villages to force those villages to remain loyal. By identifying the genetic relationships between these women, we may find evidence to support or refute this hypothesis. A determination of A, B, C, D, and X haplogroups for these samples will also show its relationship to modern populations and will be compared to other ancient samples.
For this ancient genetic analysis, mitochondrial DNA (mtDNA) will be the nucleic acid studied. A ring of about 16,500 base pairs, the mtDNA genome is strictly maternally inherited and found in the mitochondria of each cell (1). The increased number of copies of this molecule per cell is beneficial in that more copies are likely to have survived the degradative processes that ancient samples undergo. Another reason for the use of mtDNA in ancient genetic studies is a region on this molecule known as the displacement loop (D-loop). This is a non-coding region and therefore sustains a higher mutation rate then nuclear DNA and other regions of the mtDNA genome. The mutation rate of this D-loop is ideal for identifying individual as well as population specific polymorphisms (7).
Ancient DNA extraction has proven to be most successful on teeth and bone samples (9). These samples are usually better preserved thus containing greater amounts of DNA and are less likely to be contaminated by modern DNA. Such samples were removed from the individuals at the time of excavation and 15 of those samples have been brought to the Molecular Archeogenetic Research Laboratory at Brigham Young University. Extraction methods included a .5 M EDTA digestion of .1-.5 g of powdered material (3). The powdered material was obtained through drilling into or crushing parts of the sample. After digestion, the DNA was retrieved through an ion column or binding to glass beads (10). Extraction procedures were performed on all fifteen samples brought into the lab.
The degraded nature and limited amount of DNA in ancient samples makes them very susceptible to modern contamination. Strict methods must be employed in the field at the time of excavation to maintain the integrity of the sample. Many precautions as prescribed by others (5,7) are taken to prevent contamination from happening in the laboratory. The surface area of the sample is cleaned extensively or avoided during the extraction process. The entire extraction process was done in a restricted access laboratory dedicated strictly to the isolation of ancient DNA. Hoods and lab benches used were irradiated with ultraviolet light to eliminate any contaminating DNA. Negative controls were processed concurrently with all extraction and amplification processes to monitor possible contamination and identify any false positives.
Once the DNA was extracted it was exposed to a series of Polymerase Chain Reactions (PCR). The first is a PCR based procedure that repairs nicks, breaks and other DNA damage (2,10). Hypervariable regions I and II of the D-loop region of the mtDNA will then be amplified through primer specific PCRs (5,8). Following amplification the DNA is sequenced using automated sequencing (10). From the resulting sequences, relationship matrices can be produced. From these matrices, obvious maternal and other relationships can be established.
Sequences were obtained for six of the samples tested. These sequences yielded about 110 bases of information from Hypervariable region I from (base numbers 16180-16298) of the D-loop of the mitochondrial DNA. When aligned and compared these sequences show a lot of similarity. The following graph shows the few changes in bases found in the sequences studied.
The similarity between these sequences indicates a close maternal relationship between these individuals. If this were true it would disprove the theory that these women were taken from different villages. It would support the idea that they are all related and probably part of the royal household. The similarity of these sequences may also indicate contamination. To verify these results the samples need to be tested again and a second sequence obtained. If the second sequence matches the first then it is less likely to be contaminated. Once the sequences have been verified the analysis will prove more informative. Father analysis would include a haplogroup comparison to determine the haplogroups of these individuals. These could then be compared to other populations.
References
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- Woodward, S. R., King, M. J., Chiu, N. M., Kuchar, M. J., Griggs, C. W. (1994) PCR Methods and Applications 3:244-247
- Woodward lab protocols