Extração de DNA antigo de populações nativo-americanas: um estudo pioneiro baseado em protocolos internacionais

Abstract

Ancestral DNA (aDNA) refers to the genetic material extracted from archaeological remains, which has been extensively studied in human evolution and various other genetic investigations over the past 40 years. To obtain aDNA, a bone fragment or tooth is pulverized and decalcified, followed by isolation and purification of the DNA, often supported by methods such as polymerase chain reaction (PCR) and Sanger sequencing to increase the yield of extracted aDNA. Despite international advances, this field has seen little development in Brazil, with no records of extractions performed exclusively within the national territory. Therefore, the present study aimed to describe the first protocol adapted for aDNA extraction conducted entirely in Brazil using an experimental method in a BSL-2 laboratory. To that end, a sterile environment was prepared for the pulverization of sixteen tooth samples from the pre-colonial period. Subsequently, the extraction and purification of this material were carried out using the automated Extracta 32™ system, which employs a magnetic bead-based method. The quantification and qualification of the extracted DNA were evaluated using a NanoDrop™ spectrophotometer. The hypervariable regions 1 (HVR-1) and 2 (HVR-2) were then amplified using PCR. Finally, Sanger sequencing was employed for initial screening of mitochondrial haplogroups present in the amplified samples, aiming to support future library preparation for next-generation sequencing (NGS). All samples showed detectable concentrations of aDNA, ranging from 8.3 ng/µL to 32.9 ng/µL, a variation consistent with previously published data. Moreover, the mean A260/280 absorbance ratio was 1.64, slightly below the ideal range of 1.8, which may reflect the fragmented and degraded nature of the extracted aDNA. Additionally, most samples were successfully amplified for at least one of the HVRs, and Sanger sequencing identified Native American haplogroups (A, B, C, or D) in eight samples. Therefore, the results obtained in this study suggest that the workflow used was effective for retrieving aDNA, which will be further explored in a subsequent study involving more robust analysis through NGS.