Comparison of bone demineralisation procedures for DNA recovery from burned remains

Recovering DNA from modern incinerated bones can be challenging and may require alteration of routine DNA extraction protocols. It has been postulated that incinerated bones share some similarities with ancient bones, including fragmented DNA, surface contamination and highly mineralised structure, all of which can inhibit the successful recovery of genetic material. For this reason, ancient DNA extraction protocols are often used for incinerated modern samples; however, their effectiveness is still somewhat unclear. Much of this uncertainty exists around the demineralisation step of extraction, specifically the length of incubation and retention or removal of supernatant. As obtaining human samples for forensic research can be challenging, porcine models (Sus scrofa domesticus) are often used as substitutes. This study developed real time PCR assays for porcine nuclear DNA in order to investigate the effects of modified demineralisation protocols on DNA yield from femurs exposed to either short (60 min) or prolonged (120 min) burning. Gradient PCR results indicated 56 °C was the ideal amplification temperature for targeted amplicons, with melt curve analysis showing short and long amplicons corresponded to 80.3 °C and 83 °C peaks respectively. Results of altered extraction protocol showed a trend towards higher DNA yields from longer demineralisation periods however this was not significant. By comparison, retaining supernatant post-demineralisation resulted in significantly greater DNA yields compared to discarding it (P < 0.009). Although DNA content yield decreased with burn duration, the demineralisation treatment variations appeared to have the same effect for all burn lengths. These results suggest that for incinerated modern bone retaining the supernatant following demineralisation can dramatically increase DNA yield.