**3. Examples of ancient DNA extraction procedures**

An example of DNA extraction from a pre-Hispanic sample is depicted in Figures 1 and 2. Figure 1 displays the contaminants with different colours during the phenol-chloroformisoamyl alcohol procedure. Figure 2 shows the DNA extracted by the phenol-chloroformisoamyl alcohol technique from 0.25 g of two powdered bone samples from the same individual. In this figure, we observe the sample contaminants that are one of the major

All amplifications were carried out in a GeneAmp® PCR System 9700 thermocycler with the following profile: 5 min at 94ºC, followed by 40 cycles of 1 min at 94ºC, 1 min at 59ºC (haplogroups A, D and RHVs) or 55 ºC (haplogroups B and C), and 1 min at 72°C, with a final extension of 10 min at 72ºC. At least one PCR blank was amplified alongside each

The PCR products were visualised on 2% agarose gels with ethidium bromide, and all positive products were purified using the QIAquick kit (Qiagen) and sequenced using the BigDye® Terminator v3.1 kit (Applied Biosystems) in an ABI PRISM 310 genetic analyser.

Phylogenetic analysis. The sequences of the pre-Hispanic PCR products from the HVI segment were aligned with representative Amerindian mtDNA control-region sequences (GenBank accession numbers: AY195760, EU719927, EU719811, EU719679, EU720004, EU720308, EU720078, EU719797, AY195749, EU720177, EU720123, EU719764, HQ012155, EU720242, HQ012184, HQ012164, HQ012134, AY195772, EU720073, EU720339, AY195759, EU720071, EU720336, EU720102, EU720202, HQ012188, HQ012198, EU720029, HQ012255, HQ012254, HQ012253, GQ449339, EU034320, AY195748, AF214088, DQ973581, AF478614) and two ancient sequences from a prehistoric Oneota population (Stone and Stoneking, 1998) using the Clustal W program (Thompson et al., 1994). Then, the phylogenetic tree was constructed with the Jukes-Cantor method, and the distances were obtained from a neighbour joining algorithm. Finally, the tree was optimised for maximum likelihood, using

Haplotype network analysis. The median-joining (є=0) networks (Bandelt et al., 1999) of haplotypes were constructed using the Network package, v4.5.1.0 (Fluxus Engineering). Sequences used were those described for phylogenetic analysis. This method is for

An example of DNA extraction from a pre-Hispanic sample is depicted in Figures 1 and 2. Figure 1 displays the contaminants with different colours during the phenol-chloroformisoamyl alcohol procedure. Figure 2 shows the DNA extracted by the phenol-chloroformisoamyl alcohol technique from 0.25 g of two powdered bone samples from the same individual. In this figure, we observe the sample contaminants that are one of the major

H712-730 5'-CCAGTGAGTTCACCCTCTA-3' (Parr et al., 1996).

H8297-8316 5'-CTGTAAAGCTAACTTAGCAT-3' (Wrischnik et al., 1987);

H13384-13403 5'- ATATCTTGTTCATTGTTAA -3' (Lorenz and Smith, 1996)

H5230-5249 5'-TGCCCCCGCTAACCGGCTTT-3' (Stone and Stoneking, 1993)

L8196-8215 5'-ACAGTTTCATGCCCATCGTC-3';

L13198-13213 5'- GCAGCAGTCTGCGCCC -3';

L5101-5120 5'-TAACTACTACCGCATTCCTA-3';

Hy-Phy software (Kosakovsky-Pond et al., 2005).

constructing networks from recombination-free population data.

**3. Examples of ancient DNA extraction procedures** 

Haplogroup B:

Haplogroup C:

Haplogroup D:

**2.4 Data analysis** 

(1996)

batch.

obstacles to these studies because they inhibit the Taq polymerase. The contaminants, such as Maillard products of reducing sugars (Pääbo, 1989) and humic acids with phenolic

groups, were observed by fluorescent stain in blue while the DNA degraded (which results in a smear pattern) is stained in pink by ethidium bromide (Figure 2, panel A). Figure 2, panel B shows the second sample in lane 3, which displays only contaminants. The DNA was not apparent. These compounds can be partially eliminated using kits such as the Amicon® Ultra-0.5 30 kDa columns. These results show variation in DNA yields between extracts taken from different samples of the same bone, even when using the same extraction method. We attribute such differences to heterogeneity within the bones.

Fig. 1. Extraction of aDNA of bone samples from four different pre-Hispanic samples using the phenol-chloroform-isoamyl alcohol technique.

Fig. 2. Extraction of DNA of two independent samples (A, B) from the same pre-Hispanic individual by the technique of phenol-chloroform-isoamyl alcohol and ethanol precipitation. Lanes 1, molecular weight markers of *HindIII*; Lanes 2, no-sample; lane 3, DNA extracted from sample 1 of the Mexican pre-Hispanic population from Monte Alban.

Extraction and Electrophoresis of DNA from the Remains of Mexican Ancient Populations 487

Fig. 4. Inhibition of the mitochondrial DNA hypervariable segment I amplification via

Fig. 5. Positive effect of bovine serum albumin (BSA) on the PCR performance of aDNA

Sometimes aDNA dilution is not enough to obtain the PCR products, so we tested the effect of BSA addition by increasing the concentration of BSA in the reaction from 0.1 to 0.25 mg/ml. Figure 5 shows the positive effect of BSA on the PCR of aDNA extracted by the phenol-chloroform-isoamyl alcohol procedure. Increased amplification was observed in the PCR experiments when BSA was added in increasing concentrations (Figure 5, lanes 2-7, BSA at a concentration of 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3 mg/ml, lane 8, negative control and

extracted by the phenol-chloroform-isoamyl alcohol procedure.

inhibition of Taq polymerase by aDNA contaminants.

Because DNA concentration is only possible with limited precision and concentrations of standard dilution series change over time in storage, we evaluated the relative performance of the DNA during PCR amplification using serial dilutions of the extracted DNA starting from 5 μl. Using this method, we were able to dilute the inhibitors of the Taq DNA polymerase.

Fig. 3. Extraction of DNA of pre-Hispanic samples by the silica technique. Lanes 1 and 10, molecular weight markers of 23 kbp and 100 bp, respectively. Lanes 2 to 9, DNA extracted from different samples of Mexican pre-Hispanic populations.

Silica gel was also used to purify aDNA, results are shown in Figure 3. Each lane of this figure (2-9) displays aDNA extracted from 0.25 g of different pre-Hispanic bone samples. Lanes 1 and 10 show molecular weight markers. The quantity of Taq polymerase inhibitors is not evident, although we know that all ancient samples contain some of these inhibitors in different concentrations.
