Molecular Archaeology

Different methods have been used and are being used in the analysis of archaeological data. Among others, different archaeometric fields such as paleoecology (paleozoology, paleobotany and pllenanalysis), dating methods (radiocarbon-dating and dendrochronology) and analytical chemistry had been used for the evaluation of the quantity and quality of different archaeological substances (Kiesslich, n.d.). Given the nature of archaeology, evaluated data are analyzed on the point of view of history. The recent discoveries in science particularly in genetics and molecular biology have given rise to another method of scientific analysis of archaeological data. The new developments allow easier investigation of ancient remains not only through paleoecology, dating and chemical methods but on a molecular level. This new branch of archaeological analysis is what is now known as Molecular Archaeology. Christianson (2007) of the Minnesota State University gives a more perspicuous description of the field. According to him, Molecular Archaeology is an â€Å"†¦emergent field in archaeology that has been brought about by the advancements of the recognition and understanding of DNA, focusing on the acquisition of either DNA or mtDNA (mitochondrial DNA) and being able to determine species of natural archaeological finds as well as determine blood lines and/or sex of animal or human remains.† It is this use of the DNA in the analysis of archaeological data that differentiates Molecular Archaeology from the other methods of archaeological analysis. It is the biological function of this DNA that makes molecular archaeology an irreplaceable field in analytical archaeology. This DNA contains genetic information which, once known, could provide special information about the individual properties of the probe (Christianson, 2007). These individual properties include one's species, population, and gender. Also, Through the use of DNA residues, accuracy in reconstructing subsistence and related cultural activities is more probably. As a result, we gain more knowledge and understanding of the lives of our ancestors and the environment they lived in as well as of the other creatures that coexisted with them (Christianson, 2007). There was a time when molecular archaeology seemed to be inconceivable. This was when scientists believed that DNA-preservation was impossible in biological remains. Previous studies have shown that it only took days or even hours for degradation to occur after the death of an individual. With degradation, of course, is the loss of significant genetic information (Keisslich, n.d.). It is one study in the early eighties defied this scientists' limiting belief on the relationship between degradation and DNA-preservation and paved the way for molecular archaeology and the use of DNA in the analysis of archaeological data. â€Å"This is the successful detection of intact genetic information in a 4000-year-old Egyptian mummy† (Kiesslich, n.d.) Furthermore, the invention of analytical methods in molecular biology boosted the field of molecular archaeology. â€Å"In 1988, Mullis and Saiki published a study on Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase†. Practically, they invented the PCR or the Polymerase Chain Reaction technique which made possible the detection and characterization of even minimal traces of DNA. In theory, the presence of even a single intact molecule of DNA can give a positive result (Kiesslich, n.d.) †The impact of molecular archaeology particularly its use of DNA in evaluating data has been proven to be a great leap in archaeological research†. Because DNA is a huge aspect of molecular archaeology, it is necessary even for the general studies of this subject to include information on the nature of the DNA. DNA is a helically-twisted macromolecule consisting of a sugar-phosphate backbone. â€Å"Each sugar in the DNA's sugar-phosphate backbone is bound to one side-chain which may be different for each unit†. It is this side-chain that represents a single basic unit of DNA or DNA-base. It is the connection between a few to several billions of base-pairs connected by one polymer unit that produces a double helix, particular to the DNA. (Kiesslich, n.d.) The function of the DNA is mainly the maintenance and passing of genetic information from parent to progeny. This genetic information is encoded in 4 different letters (A, C, G, T) which represent the bases or the basic units. Three of the letters together correspond to the next superior information unit (Kiesslich, n.d.) The DNA not only functions for the maintenance and passing of genetic information but also for the control of the biological functions of each cell. For living organisms, it is located in the nucleus of the cells and in small cellular compartments called mitochondria. This mitochondria are considered as the powerhouses of the cell because it provide for the energy needed for cellular processes. Like nuclear DNA, mitochondrial DNA has their own chromosomes. â€Å"In general, a cell contains only two copies of nuclear DNA and as much as a thousand copies of mitochondrial DNA â€Å"(Kiesslich, n.d.). What is crucial to the nature of the DNA is the sequence of its 4 discriminable bases or the base-sequence. It is this base-sequence that represents the genetic information passed on from parent to progeny and controls the cellular chemical reactions. It is this sequence that is the targeted information in the analysis of archaeological facts and this sequence can only be determined through the application of methods in molecular biology (Kiesslich, n.d.). Information given by an individual's DNA is not limited to the individual but also to pathogens which include microorganisms and bacteria, as well as biological materials and the diet of a settlement (Kiesslich, n.d.). DNA of microorganisms and bacteria can be determined through paleopathology and paleoepidemiology. DNA of biological material can be determined through paleoecology, paleobotany and paleozoology. An extracted intact DNA would give the whole genetic information of an individual. This genetic information can be accessed using different sequence-specific DNA probes which also provide information required for archaeological analysis (Kiesslich, n.d.) There is a difference between the extraction of DNA from the nucleus and DNA from the mitochondria. Note that DNA from the nucleus persists only as two copies for every cell while mitochondrial DNA persists at an average of a few thousand copies per cell. â€Å"Nuclear DNA and mitochondrial DNA require a minimum state of preservation to obtain the specific sequence information†. The greater quantity of mitochondrial DNA allows it to be more readily accessible compared to nuclear DNA. It is also more resistant to degradation processes after death as well as diaganetic influences compared to nuclear DNA (Kiesslich, n.d.). The two types of DNA are different in the types of information that can be obtained. In nuclear DNA, information about an individual's genetic constitution can be obtained. This includes the individual's species, gender, fingerprint and kinship with other individuals. Meanwhile, the mitochondrial DNA provides information that allows the assessment of maternal lineage within a community. This is because mitochondrial DNA is exclusively maternally hereditary. The mitorchondrial DNA's comparatively slow mutational rate also allows greater evaluation of genetic context for individuals. In effect, individuals can be classified through middle and long-range temporal classification (Kiesslich, n.d.). Using information on the X and Y chromosomes, gender can be determined easily using molecular biological methods (Faerman and Filon, 2005). As it is known, gender is one of the most significant features of an individual and is likewise significant in archaeological analysis. Among other parts, gender can be and is usually determined using the teeth and the bones (Kiesslich, n.d.). Sex-specific genes are located in the X and Y chromosomes. Still, the determination of gender is not limited to molecular biological means. The determination of the gender of an individual remains can be done through conventional methods including morphometry. This happens when convenience is not the issue but the quality of the sample itself when it is too damaged or when the remains that are analyzed belonged to an infant. As a rule, a few grams of bone or tooth is enough for DNA-analysis (Kiesslich, n.d.). †Gender determination through DNA-analysis can be exemplified by the study done in South Israel, at a bath house at a burial site of Roman Askalon†. It must be noted that in Ancient Askalon infanticide was a widespread phenomenon (The Advent of Molecular Archaeology, 2005). †Behind the bath house, archaeologists found more than 100 skeletal remains of infants which at first were thought as the remains of unwanted girls†. The epigraph stating â€Å"Enter, Enjoy and†¦Ã¢â‚¬  and the several lamps with erotic motifs gave rise to a theory which was confirmed using DNA-analysis. Through gender determination by DNA-analysis, it was found out that the skeletal remains that were found did not only belong to unwanted girls but also to unwanted boys. The bath house was confirmed to be a brothel and the skeletal remains were the infants of the women who used to work at the place (Kiesslich, n.d.). Meanwhile, the issue on whether or not tuberculosis was brought to Peru by Columbus and his successors was clarified using DNA analysis. â€Å"One study reported that the DNA of tuberculosis pathogens already existed even among 600-900-year-old Peruvian mummies† (Kiesslich, n.d.). Nuclear DNA-analysis is also used in the identification of remains. In history, the remains of Josef Mengele in Brazil as well as the identification of the remains of the Romanov family in Jekatrinenburg after the Bolshevik Revolution were identified using DNA-fingerprinting (Kiesslich, n.d.). The analysis of organic residues in some jars found in Egyptian Amphorae allowed the discovery of what commodities were transported to Egypt during the Late Bronze Age and the links between the sources of the jars, the commodities and the way of transport of substances in the Mediterranean world (Stern, 2001). Mitochondrial DNA analysis was used in the investigation of the Tyrolean Ice-man (The Advent of Molecular Archaeology, 2005). The findings of the investigation revealed a high DNA-sequence homology to today's population in the Northern alps (Kiesslich, n.d.) and showed a great fit to the Northern European context. In this case, clothes and tools associated with the findings were also investigated aside from the individual body, giving an idea on vegetation during the era (Kiesslich, n.d.). DNA-analysis also covers topics historical and anthropological topics particularly population-genetics. Population-genetics include the tracing of migrations and distributions of populations. For example, kinship analysis was done with some individuals in Forida (Kiesslich, n.d.). DNA-analysis can also be used to trace genealogical origins and also in the determination of possible threats of diseases from ancestors. Source materials for DNA-analysis are not limited to bones and teeth. As a matter of fact, anything that could possibly contain DNA, even if not part of the individual's body can be a source material. â€Å"Source materials can range from biological remains such as skeletons, bodies, bones, hair, teeth, forensic and medical preparations, museum specimens, fossils and objects that an individual has come in contact with† (Kiesslich, n.d.). It must be remembered that DNA-analysis is a procedure which involves the destruction of the specimen. This implies that once a specimen has been used for analysis, it cannot be reused. On the other hand, even small amounts of materials, say, a piece of hair or a gram of bone is enough for any DNA-analysis as long as the specimen is of quality, depending on the source's chemical, physical, geological, ecological and biological history (Kiesslich, n.d.). †A DNA can be expected to be intact and well-preserved if it has been maintained at low-temperatures such as the case of the Tyrolean Ice-man, or if it has been maintained in arid places†. As a rule, DNA-degradation happens under humid conditions so specimens coming from deserts, and in polar regions or any other setting with similar conditions would produce more intact DNA specimens and more successful DNA analysis. Other conserving factors include anaerobic conditions such as that in Florida during the kinship analysis and the quantity of possible DNA specimens such in mummies (Kiesslich, n.d.). The less a specimen is affected by diagenetic processes, the more intact and well-preserved it will be when used for DNA-analysis. This is the reason why teeth and bones are commonly used for DNA-analysis. Their structure, and rigidity as well as the little hollow spaces with single cells, which undergo individual post-mortem mummification (Kiesslich, n.d.). In addition, these specimens are less affected by natural contamination during the life of the individual as well as post-mortem contaminations. Contemporary contaminations in the specimens can also be easily removed before extracting the DNA. As noted earlier, teeth and bones are suitable material sources for the determination of gender and for any other DNA-analysis (Kiesslich, n.d.). In molecular archaeology as well as in any other field that require genetic analysis, collection of samples and pre-treatment require maximum precautions. This is to prevent contemporary errors which are possible sources of errors. Errors are especially crucial in DNA-analysis since specimens are not infinite (Kiesslich, n.d.). One simple precaution is to wear gloves and safety-masks. Also, tools and containers that will be used in the analysis should be sterile and free from other DNA contaminants. It must also be remembered that probing of specimens should be done directly at the excavation site, sealed and only opened until it get to the laboratory. All these should be obligatory to prevent contamination and thus, errors (Kiesslich, n.d.) There are many procedures for the extraction of DNA from material sources. Naturally, speciments are cleaned first to remove physical contaminants such as soil and dirt. Specimens are then homogenized and placed in an extraction buffer. This extraction buffer contains compounds that are necessary for the breaking of the source-matrix. The breaking of the source matrix is done by decalcification or and proteinase-digestion. The end product is the extracted solubilized DNA (Kiesslich, n.d.). Similar to extraction, isolation and purification are also done through different procedures. Before doing the process, the chemical and physical conditions of the source material must first be evaluated. This is another preventive procedure to minimize errors from contaminants. Once the DNA in the source material has been purified and contained in an aqueous solution, substances that are co-extracted with the DNA including humic acids and other chemicals which have similar properties with the DNA must be removed to avoid false negatives in PCR reactions. These co-extrants could also inhibit enzymatic reactions (Kiesslich, n.d.). The most powerful tool for the investigation of DNA is the Polymerase Chain Reaction (PCR) because of its sensitivity which allows even a single intact DNA enough for detection. The PCR is an amplification procedure that is sequence specific. â€Å"Here, sequence specific DNA probes are added to reactions considering reaction parameters necessary for the process†. Through this, the target sequence is amplified until detectable amounts are obtained. However, further care must be done in order to avoid contamination, particularly contemporary ones. Contemporary contaminants are better preserved in the PCR. To manage this, blank extracts and zero-controls must be done for every extraction. In sum, controls are necessary to for the verification of the authenticity of the results and in order to trace possible contaminations that are present (Kiesslich, n.d.). Zero-controls are PCR-reactions which do not contain the DNA being analyzed (Kiesslich, n.d.). They are blank extracts which contain everything that is required for the reaction used in the DNA-analysis such as solutions and buffers. In the same way as the source material, these controls undergo the same extraction steps. Much has been transformed by the discovery of DNA. In the past, archaeological investigations may be limited to the physical level. Today, it has reached the era when Archaeology intersects with Molecular Biology. There are still lots of evidences to discover. history is still filled with gaps. With the advent of Molecular Archaeology, strands may just be made and gaps may just be filled. References Christianson, B. (2007). Molecular Archaeology. Minnesota State University. Retrieved 23 October 2007 from http://www.mnsu.edu/emuseum/archaeology/archaeology/moleculararchaeology.html. Faerman, M., D. Filon, et al. (1995). Sex identification of archaeological human remains based on amplification of the X and Y amelogenin alleles. Gene, 167, (1-2): 327-32. Kiesslich, J. (n.d.). The Emerging Field of Molecular Archaeology. Retrieved 23 October Saiki, R. K., D. H. Gelfand, et al. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science,   239, (4839): 487-91. Stern, B. (2001). Organic Residues in Egyptian Amphorae. University of Bradford. Retrieved 23 October 2007 from http://www.brad.ac.uk/acad/archsci/depart/resgrp/molarch/egypt.html. The Advent of Molecular Archaeology. (2005). Retrieved 23 October 2007 from http://humanitieslab.stanford.edu/2/184.