**5. Conclusion**

494 Gel Electrophoresis – Advanced Techniques

from museums or collections where the researcher does not know how they were managed. In these conditions, additional controls are recommended for all of the

Fig. 12. Phylogenetic analyses of American native populations including five sequences from samples of pre-Hispanic populations. Tree of Native American and ancient pre-Hispanic Amerindian, constructed with the Jukes-Cantor method, and the distances were obtained from a neighbour joining algorithm and optimised for maximum likelihood using Hy-Phy software (Kosakovsky-Pond et al., 2005). The lanes in different colours indicate the haplogroup designation of lineages. Sequences of this study are marked with an asterisk.

procedures.

This review offers a direct overview of the different methods of aDNA extraction, including all special conditions needed in the laboratory to avoid contamination by contemporary DNA. It reveals the complexity involved in demonstrating the authenticity of human aDNA because the risk of contamination is very high. However, exogenous DNA contamination can be avoided if the necessary care is taken. In our experience and the experience of other laboratories, obtaining the ancient sample with coat, gloves and mask, and maintaining it in sterile conditions without human contact reduces the chances of sample contamination. It is also very important to test all reagents to verify that they are free of contemporary DNA. In addition, we also recommend performing negative control PCR experiments with at least 45 cycles to convincingly demonstrate the absence of contemporary DNA contamination. In our experience the best method to purified aDNA is phenol-chloroform-isoamyl alcohol with concentration using Amicon® Ultra-0.5 30 kDa columns (Millipore, Billerica, USA) or the Silica gel method using the QIAquick (Qiagen) columns. We also prefer to include the EDTA in the extraction buffer to optimise the aDNA extraction. This is supported by recent

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publications that have demonstrated that some DNA may be lost during decalcification (Campos et al., 2011). It is also important to keep DNA at -70°C in aliquots to maintain its integrity. Maintaining bone tissue samples at -70°C during aDNA extraction is useful to avoid additional DNA degradation. In our point of view, the best method will be that containing the least sample manipulation because this will avoid DNA contamination. Finally, there will be always risk of contamination by contemporary human DNA; however, next generation sequencing methods do provide a greatly improved means of measuring the degree of contamination in a sample.

Sequencing of the PCR products from aDNA as well as phylogenetic and network analyses of remains from America would allow testing of the hypotheses concerning single versus multiple waves of migration to the New World. This analysis will also reveal new haplotypes that were lost through time because not all migrations were successful in terms of leaving descendants among contemporary populations. Furthermore, the development of next generation sequencing is revolutionising aDNA research. The examples presented in Figure 13 and 14 display the relationship between the Oneota sample and that from Teotihuacán showing different haplotypes. There were also two ancient samples from Monte Albán that were grouped with the more frequent haplotype in the D1 haplogroup. Further analysis of more pre-Hispanic human samples will give us more detailed information about the history of these populations.
