Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Abstract

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

Long term conservation of DNA at ambient temperature. Implications for DNA data storage

“…This allows to calculate the time necessary for a DNA molecule to degrade down to 25 nucleotides, the average length which is the current limit for the sequencing of degraded DNA [ 31 ] using the formula: …”

Forensic proteomics

“…Highly degraded samples may still, theoretically, pose a challenge if DNA template levels become too low. The sensitivity levels at which sex-determination using forensic DNA methods begin to fail appears not to be demonstrated, although this has been demonstrated using ancient DNA methods that are more sensitive [258,[351][352][353][354]. Proteomic sex estimation outperforms both osteological and ancient genomic methods [258].…”

“…Proteomic sex estimation is a validated technique with clear applicability in archaeological contexts [353,354]., the utility in forensic contexts needs to be established. Any new method requires rigorous validation tests to assess the overall performance, advantages, and possible caveats, in order to evaluate its range of applications within forensic workflows.…”

Long term conservation of DNA at ambient temperature. Implications for DNA data storage

“…This gave a degradation rate constant of 3.82x10 -15 cuts /s/nucleotide, equivalent to about 1 cut/century/100 000 nucleotides or 38 000 years of half-life for a 150-nucleotide long DNA fragment (we chose this size for an immediate comparison with the previous works [28,29]). This allows to calculate the time necessary for a DNA molecule to degrade down to 25 nucleotides, the average length which is the current limit for the sequencing of degraded DNA [31] using the formula: So, it appears that the DNA stability at room temperature (25 °C ) is over three orders of magnitude higher in DNAshells than in any of the other currently commercialized storage devices. This is to be expected because, first, FTA paper, trehalose or calcium phosphate leaves the DNA samples directly exposed to the atmosphere.…”