**Extraction and Electrophoresis of DNA from the Remains of Mexican Ancient Populations**

Maria de Lourdes Muñoz et al\*

*Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico D. F. Mexico* 

#### **1. Introduction**

478 Gel Electrophoresis – Advanced Techniques

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Ten years ago, the first reports of human genome sequencing were published in Nature and Science (Venter et al., 2001; Sachidanandam et al., 2001; Lander, 2011). This was very exciting and expectations for the application of genome sequencing technology were high. In the past decade, the cost of sequencing has gone down several orders of magnitude, making it a more accessible technology for research studies. The medical value of comprehensive genome sequencing is now becoming apparent: for example, the genetic cause of a rare and debilitating vascular disorder was solved by genome sequencing at NIH (Jasny and Zahn, 2011; Lander, 2011). It is also possible to solve the genetics of individual Mendelian disorders thereby relating phenotype to genotype. In addition, better treatments for diseases such as cancer, metabolic disorders, inflammation, neurodegeneration or diabetes are expected to be found through studies involving genome sequencing (Lander, 2011). Sequencing also has been used to query variation in populations worldwide, and sequences are now available from extinct hominids as well as from thousands of other species (Rasmussen et al., 2010; Krause et al., 2010; Reich et al., 2010; Balter, 2010; Rasmussen et al., 2010). We expect to know very soon what variation exists among individuals at almost all sites in the genome. This is a great opportunity for population genetics to reconstruct the entire genealogical and mutational history of humans (Callaway, 2011), to understand the evolutionary and genetic forces that affected every region of the genome, to determine disease mutations present in human populations, to elucidate the genetic bases of cognitive and physiological adaptations, and/or to determine the demographic events that led to the colonisation of the earth.

The question remains: what is the relationship between morphological features and ancient deoxyribonucleic acid (aDNA)? The evolutionary processes that generated modern species

<sup>\*</sup> Mauro Lopez-Armenta1,2, Miguel Moreno-Galeana1, Alvaro Díaz-Badillo1, Gerardo Pérez-Ramirez1, Alma Herrera-Salazar1, Elizabeth Mejia-Pérez-Campos3, Sergio Gómez-Chávez4 and Adrián Martínez-Meza5

*<sup>1</sup>Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Mexico* 

*<sup>2</sup>Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México, Mexico* 

*<sup>3</sup>Instituto Nacional de Antropología e Historia, Querétaro, Mexico D.F., Mexico 4Teotihuacan, Mexico* 

*<sup>5</sup>Mexico City, Mexico* 

Extraction and Electrophoresis of DNA from the Remains of Mexican Ancient Populations 481

population genetic studies will be discussed. Technical differences between DNA extraction procedures for ancient bones and mummy tissue will also be described. Molecular phylogenetic analysis, haplotype and haplogroup determination through software will also

There have been several aDNA extraction protocols suggested over the years. The first method was purification based on phenol/chloroform extraction, alcohol precipitation (Kalmár et al., 2000; Munoz et al., 2003; Hagelberg and Clegg, 1991; Hänni et al., 1995) and silica binding (Höss and Pääbo, 1993; Yang et al., 1998). In addition, other methods have been suggested, such as using Chelex (Faerman et al., 1995), centricon filters (Anzai et al., 1999), Dextran Blue (Kalmár et al., 2000), decalcifying bone with EDTA (Hagelberg and Clegg, 1991; Hänni et al., 1995; Yang et al., 1998) and hybridisation and magnetic separation (Anderung et al., 2008). The methods most commonly used now combine EDTA decalcification and silica purification (Yang et al., 1998; Krings et al., 1997; Anzai et al., 1999). It is evident that many different techniques have been used, demonstrating that no single procedure has clear advantages. Based on our experience, the selected method is a function of the sample characteristics, including considerations for the origin of the sample, from the

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

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).

All DNA purification and PCR experiments were carried out under sterile conditions in separate dedicated rooms. Samples were handled wearing protective clothing from collection to DNA isolation, and the laboratory equipment and reagents are maintained DNA-free. The laboratory managing the ancient samples has a high-pressure system to filter the incoming air and a laminar flow hood as well as UV light irradiation and bleach were

used to clean of every surface to avoid contamination (Knapp et al., 2011).

requirement of ethics review by the Research Ethics Boards.

be defined and examined.

skeleton or a mummy.

**2.2 Ancient DNA extraction** 

**2.1 Samples** 

**2. Procedures to study ancient DNA** 

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 Denisovans (Reich et al., 2010).

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 the human population.

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, taking into account ancient mtDNA studies.

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
