**2.1 Samples**

480 Gel Electrophoresis – Advanced Techniques

and populations are commonly inferred through the analysis of morphological and genetic markers in addition to analyses of contemporary organisms to create tentative reconstructions. To confirm this indirect evidence, it is necessary to check the reconstructions against the fossil records. Nevertheless, the comparison has been made possible now by analysing morphological characters, and the application of recent advances in deoxyribonucleic acid (DNA) sequencing technologies for aDNA are now allowing the genetic record to be generated. This new technology let us focus not only on single genetic loci, such as mitochondrial DNA (mtDNA), but it made possible to obtain whole genome sequences of extinct species and populations (Lander et al., 2011), our closest extinct relatives the Neanderthal (Green et al., 2010), and the extinct hominid group from Siberia,

The field of aDNA was initiated more than twenty years ago (Higuchi et al., 1984; Cooper et al., 1992; Greenwood et al., 1999) and research efforts continue to grow and expand into new areas (Stoneking and Krause, 2011). The first aDNA studies demonstrated the inefficiency of bacterial cloning to amplify small sequences recovered from the skins of animals and human mummies (Higuchi et al. 1984; Pääbo, 1985) and showed that DNA was at very low concentrations of short damaged fragments. However, these studies are considered very important because they will elucidate population origins, migrations, relationships, admixture and changes in population size, essentially revealing the demographic history of

It is now accepted that DNA is preserved in ancient samples under a wide range of depositional environments (Willerslev and Cooper, 2005). Although the DNA of a deceased organism degrades rapidly, part of it may survive for more than 100,000 years under favourable conditions, such as cold, stable temperatures and a dry environment (Pääbo et al., 2004). Fortunately, the development of new technologies has made possible the recovery and manipulation of these molecules as well as the genetic characterisation of these samples. Because this DNA is degraded the analysis is complicated, nevertheless, the new sequencing technology makes it possible to obtain historical information. In addition, the presence of polymerase inhibitors makes DNA amplification exceedingly difficult. Research in this area shares a common problem with forensics and other approaches requiring analyses of museum and non-invasively collected specimens; the amount of endogenous DNA available in the samples is limited. In addition, when working with human samples it is also possible to have contamination from contemporary human DNA. Careful adherence to currently established procedures is necessary to avoid such contamination (Deguilloux et al., 2011). Because aDNA contains the information of our past its analysis is of high importance. Here, we will review a variety of methods for extraction, purification, amplification and sequencing of aDNA segments informative for genetic population studies. Future prospects for the potential direction of ancient DNA research will be discussed. Furthermore, contributions to migratory theories will also be analysed based on population diversity,

Although there is new technology to determine the sequence of nuclear DNA, we will focus on mtDNA analysis. mtDNA analysis has been very useful to extensively examine human population history throughout the world because of its relatively rapid rate of mutation, lack of recombination and maternal inheritance. Mitochondrial DNA sequence variations at the hypervariable regions HVI and HVII will be described and their importance in

the Denisovans (Reich et al., 2010).

taking into account ancient mtDNA studies.

the human population.

Samples from this study include bones pertaining to pre-Hispanic populations from different periods of time (200 to 1500 years before present). Bone samples of two individuals from Monte Albán, Oaxaca, one from Teotihuacán and a tissue portion from the mummy Pepita were used in the examples presented in this study. To work with the ancient Mexican samples, we made a written agreement with the "Intituto Nacional de Antropologia e Historia" (Mexico). Research on ancient unidentifiable human remains is excluded from the requirement of ethics review by the Research Ethics Boards.

Sampling should be conducted as soon as the bones appear in excavation, and gloves, mask and coat must be used to prevent contamination from excavators. This is not always possible because some samples were collected before these studies were initiated. Samples also have to be deposited directly in hermetic sterile tubes and frozen at -70°C. These practices prevent the introduction of contaminant DNA during the sample collection. In addition, it is also very important to manipulate the sample in a sterile clean room, to use bleach and ultraviolet light to degrade potential contaminants and to keep strict physical separation of modern DNA work from aDNA (Miller et al., 2008, Cooper and Poinar, 2000).
