**2.2 Ancient DNA extraction**

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).

Extraction and Electrophoresis of DNA from the Remains of Mexican Ancient Populations 483

phenol-chloroform-isoamyl alcohol (24:24:1) was boiled at 94 °C for 10 min with 5% Chelex-100 and centrifuged, and an aliquot of the supernatant was taken as the template for the

Analysis in Native Americans of mtDNA by PCR amplification and high-resolution restriction analysis with 14 endonucleases (Torroni, et al., 1992; Torroni, et al., 1993; Torroni, et al., 1994a,b; Richards et al., 1996) identified four major mtDNA lineages or haplogroups (A-D). These haplogroups of Asian ancestry, each defined by specific polymorphisms, together encompass 96.9 % of the mtDNA observed in modern Native Americans. Each lineage is characterized by specific mtDNA marker: the 9-bp deletion in the COII/tRNAlys region (haplogroup B); a HaeIII restriction site gain at nucleotide position 663 of the reference sequence (haplogroup A) (Anderson et al., 1981); a HincII restriction site loss at nucleotide 13259 (haplogroup C); and an AluI restriction site loss at nucleotide 5176 (haplogroup D) (Wallace et al., 1985; Schurr et al., 1990; Torroni et al., 1992; Wallace and Torroni, 1992). Sequence data indicate a correspondence between each marker and particular hypervariable region I (HVI) mutations (Horai et al., 1993; Bailliet et al. 1994). Consequently, the mtDNA amplification of the specific region has to be performed to characterize the Native Americans (ancient and contemporary) populations. Primers to amplify HVRII were also included, although we did not included any example, because

Enzymatic amplification by PCR was performed as described previously (Munoz et al., 2003; Campos, et al., 2011) using heat-resistant *Thermus aquaticus* (Taq) DNA polymerase (FINNZYMES), or Platinum® Taq High Fidelity (Invitrogen). The PCR parameters were as follows: 2.5 U of hot start DNA polymerase, 1X buffer, 2.5 mM MgCl2, 200 µM of each dNTP, 0.25 mg/ml bovine serum albumin (BSA) and 0.2 µM of each primer in a total volume of 25 µl, and 5 µl of the aDNA template. The primers used to amplify and sequence

**2.3 Amplification of DNA from pre-Hispanic samples** 

analysing the HVRII region is not as informative as the HVRI.

human mitochondrial DNA were as follow:

L15975-15996 5'-CTCCACCATTAGCACCCAAAGC-3';

L16140-16159 5'-TACTTGACCACCTGTAGTAC-3';

L15989-16008 5'-CCCAAAGCTAAGATTCTAAT-3';

L16094-16122 5'-TCGTACATTACTGCCAGYC-3';

L16190-16209 5'-CCCCATGCTTACAAGCAAGT-3';

L610-633 5'-TGAAAATGTTTAGACGGCCTCACA-3';

L8-29 5'-GGTCTATCACCCTATTAACCAC-3';

H16401-16420 5'-TGATTTCACGGAGGATGGTG-3' (Vigilant et al., 1989);

H16236-16255 5'-CTTTGGAGTTGCAGTTGATG-3' (Wilson et al., 1995);

H16380-16398 5'-CAAGGGACCCCTATCTGAG-3' (Poinar et al., 2001);

H408-429 5'-CTGTTAAAAGTGCATACCGCC-3' (Vigilant et al., 1989)

H16130-16152 5'-AGGTGGTCAAGTATTTATGGTAC-3' (Eichmann and Parson, 2008);

H16228-16248 5'-GTTGCAGTTGATGTGTGATAG-3' (Eichmann and Parson, 2008);

HVR I:

HVR II:

Haplogroup A:

PCR experiment.

DNA purification was preceded by a decontamination step to eliminate surface exogenous DNA when samples were collected and manipulated by unknown people. Each sample was washed with bleach followed by a water rinse and UV light irradiation for 30 min on each face. Some authors suggest removing the surface of the bone, however this procedure may also contaminate the inside of the bone if it has some kind of porosity. When bones were collected as soon as they appeared during the exaction with the necessary equipment to prevent contamination from excavators (i.e., gloves, mask and coat), it was not necessary to treat the sample with the decontamination steps (Deguilloux et al., 2011) and the potential to damage template or impede the efficiency of PCR was therefore avoided. Overall, there is no way to guarantee complete removal of contaminant DNA through decontamination procedures, but these practices are used to eliminate as much contamination as possible.

Bone powder was generated by grinding in a mortar with pestle until a fine powder was obtained, when bone quantities were around 1 g. When the weight of the samples was ≤ 0.5 mg, the bone sample was ground under liquid nitrogen with a sterile screw cylinder modified from those suggested by Thomas M. G. and Moore L. J., (1997). The powder (0.250- 0.500 g) was transferred into a sterile 15 ml tube and was suspended in 2 ml of extraction buffer (0.01 M Tris-HCl, 0.1 M EDTA and 0.2% SDS pH 8.0), and the tubes were capped and sealed with Parafilm. After incubation with gentle agitation for 1 h at 37ºC, 1 mg/ml proteinase K was added, and the sample was incubated at 50ºC for 2 h. A blank extraction treated identically to the experimental samples throughout the procedure was included to monitor for contamination during the DNA extraction process. Finally, the samples were centrifuged at 5,000×g for 5 min, and the supernatants were extracted using phenolchloroform-isoamyl alcohol (24:24:1) organic extraction (Maniatis, et al., 1989; Munoz et al., 2003; Hughes et al., 2006). Subsequently, the aqueous phase was concentrated by precipitation by the addition of 0.1 volumes of 3 M sodium acetate at pH 5.0 and 2.5 volumes of ethanol. After mixing, the sample was incubated at -78ºC overnight and centrifuged at 15,000 rpm for 10 min at 4°C. The supernatant was decanted, and the precipitate was rinsed with 70% ethanol. After drying the pellet at ambient temperature in a sterile area, the pellet was resuspended in 100 µl of high quality sterile water. Alternatively, the aqueous phase can be concentrated using Amicon® Ultra-0.5 30 kDa columns (Millipore, Billerica, USA), in a final volume of 40 µl.

Another method to extract the aDNA is by binding to silica: the powered sample (0.250 g) was suspended in 1 ml of extraction buffer (0.01 M Tris-HCl, 0.5 M EDTA pH 8.0) and after incubation at 37ºC for 16 h, the suspension was incubated at 56ºC for 3 h and centrifuged at 5,000xg for 2 min. The supernatant was transferred into 3 ml of binding buffer (5 M GuSCN, 0.025 M NaCl, 0.010 M Tris-HCl pH 8.0) in a 15 ml sterile conical tube and adjusted to pH 4.0 by adding 30% HCl in 25 µl aliquots. Then, the solution is passed through a QIAquick (Qiagen) silica column. The column was rinsed twice with the washing buffer (50% ethanol, 0.125 M NaCl and 0.010 M Tris and 0.001 M EDTA, pH 8.0) and dried for 15 min. Finally, aDNA was eluted from the column with 100 µl of TE buffer (0.01 M Tris-HCl, 0.001 M EDTA, pH 8).

Extracted DNA was kept in aliquots of 25 µl at -70 ºC.

Ancient DNA can also be extracted by the Chelex-100 method: Extraction of DNA using Chelex1-100 (Bio-Rad Laboratories, CA, USA) was performed with 5% Chelex-100 in sterile H2O using the protocol described by Walsh et al. (1991). Briefly, 200 μl of DNA extracted by phenol-chloroform-isoamyl alcohol (24:24:1) was boiled at 94 °C for 10 min with 5% Chelex-100 and centrifuged, and an aliquot of the supernatant was taken as the template for the PCR experiment.
