Ancient mitochondrial DNA from hair

The DNA content of hair [1,2] is typically low compared to other tissues, as hair cells undergo dehydration and catabolic breakdown of nucleic acids and organelles during keratinisation [3]. As a consequence, ancient hair specimens have not been widely used as a source of ancient DNA. However, mitochondrial DNA (mtDNA) has been extracted from degraded and old hair samples, including burnt specimens [4], 100-yearold Native American samples [5], and wool from a 9,400 year old Bighorn sheep [6]. We have investigated the potential of hair as an aDNA source by analyzing DNA survival in 12 samples which range from 60 to >64,800 years of age and their susceptibility to contamination with modern DNA. mtDNA was successfully amplified, cloned, and sequenced from 10 of the 12 hair samples following decontamination procedures (Table 1). DNA was quantified using Quantitative Real-Time PCR in a subset of the samples (Table 1). The survival of high copy numbers of 16S DNA from the 3,000 year-old Pazyryk horse hairs is consistent with the observation that DNA survives longer at sub-zero temperatures [7]. Of greater surprise was the persistence of high numbers of 16S and Control Region DNA molecules in hairs sampled from a bison mummy 14C dated to >64,800 years. This result was independently replicated and extends the time frame from which authentic DNA has been retrieved from hair by at least seven-fold, placing it on a par with the oldest authentic DNA retrieved from bones and teeth [8]. No nuclear DNA could be amplified from the bison hair, consistent with observations of modern hair samples [1,9]. It is probably significant that the bison hairs are exceedingly well preserved — the atomic carbon to nitrogen ratio (3.47) is similar to modern mammal hair [10,11] and histological analysis of the specimen demonstrates the only structural modifications to be slight cuticular loss and adherent deposits (Supplemental data). Amplifiable mtDNA was also present in all except two hair samples preserved at warmer temperatures, although at lower levels than the animal hairs. Several studies comment on the negative effect of various hair treatments (i.e., coloring/bleaching) on DNA survival [5,12,13], and it is possible that these two samples were artificially treated pre or post mortem. Hypervariable Region 1 (HVR1) sequences obtained from hair of Andaman Island specimens are consistent with previous dental studies [14]; in contrast, several hair samples attributed to Sir Isaac Newton each yielded different HVR1 sequences. The cloned PCR products indicated that each source of hair contained only a single HVR1 sequence, but one that did not match sequences from the other samples. As each hair source has been handled on numerous occasions, it seems unlikely that only a single contaminant sequence would survive per hair sample. However, we naturally cannot remove all reasonable doubts that they may still represent DNA contamination. We therefore suggest that at least three of the hair samples do not originate from Sir Isaac Newton. This hypothesis is in agreement with the conclusions of separate isotope analyses performed on the samples (A. Wilson unpublished data). Many ancient specimens used for DNA analyses are difficult, if not impossible, to decontaminate from exogenous sources of DNA [8]. Surprisingly, although the DNA extracted from each animal hair sample was tested for contaminant human HVR1 sequences, none were detected. A similar lack of contamination appeared to apply to the human hair samples; no sequence variation was observed among cloned sequences (bar that which could be attributed to post mortem DNA damage [15]), even though the specimens have been handled multiple times by multiple individuals during their conservation history. These results suggest that hairs are either impermeable to sources of contaminant DNA (e.g., human sweat), or can be easily decontaminated (e.g. by bleach) to remove exogenous DNA. It is possible that this behavior is due to the hydrophobic nature of keratin, which comprises most of the hair shaft. The low quantities of free water associated with the keratin-packed hair cells may also reduce hydrolytic damage of the DNA. Such a scenario would explain the extended and high concentrations of DNA surviving in the bison and horse hair, as well as the relatively low levels of hydrolytic damage-induced lesions among the cloned DNA. The successful amplification of high yields of uncontaminated mtDNA indicate that hair represents a useful and underutilized source of aDNA. While the recovery of nuclear DNA from ancient hair is unlikely, this limitation also has the advantage of preventing the unintentional amplification of nuclear copies of mitochondiral sequences, which have proved problematic [8]. Furthermore, a preferential use of hair (and potentially feathers and scales) for genetic analyses would minimise the destruction of valuable historical and archaeological specimens caused by sampling of teeth or bones.