The silica-binding DNA extraction method examined in this study was proven extremely effective in providing PP16 STR profiles from bones and teeth that are 3-15 years postmortem and that were exposed to a wide variety of detrimental environmental conditions (5
). Most often these have been in contact with the soil and the other decomposing bodies in mass graves. Additionally, this method, combined with STR typing, gave a 95.4% success rate on 1823 bone or tooth samples from the 2004 Asian tsunami that were typed by the ICMP (our unpublished data). The success rate of this method is substantially higher than that of initial trials on samples from former Yugoslavia mass graves extracted using standard organic methods by the ICMP (our unpublished data) or outside laboratories (18
). The current study was performed on the same bone samples using both methods to verify and expand this comparative analysis. The silica method yielded significantly higher amounts of DNA with lower levels of inhibition than the organic method.
The samples chosen for this experiment were femur samples that contain the highest amount of DNA per gram of all skeletal elements, except teeth (6
). Larger quantity of DNA isolated by the silica-based extraction technique may suggest that the digestion buffers ATL and AL are more efficient than the Tris/NaCl/SDS/50 mM EDTA organic extraction buffer at releasing DNA from bone samples. EDTA is also present in the proprietary Qiagen buffers, although the concentration is not publicly known. It has been established that full demineralization of bone powder by high concentration has a very beneficial effect on DNA recovery (9
); however, the presence of substantial quantities of bone powder after the ICMP silica extraction indicates that demineralization is not complete. Another possible reason for the larger final amount of DNA could be that the Qiagen silica membrane is more efficient in recovering the DNA than the phase separation process of the organic extraction technique.
The real time PCR quantification system Quantifiler was chosen to quantify the DNA extracted from the bone samples because it is accurate over a wide range of DNA concentrations (0.023 ng/μL to >50 ng/μL) and it is also capable of assessing the levels of PCR inhibitory compounds in a DNA extract. The silica-based DNA extraction technique described here has been shown to isolate on average three times more DNA than the organic extraction method. The samples extracted by the ICMP silica method were all quantified at 23 pg/μL or higher, while only 45% of the samples extracted by the organic method were above this level. On the whole, organic extractions also showed substantially higher levels of inhibition, with 65% of the extracts showing a one-cycle or greater increase in the Quantifiler Ct values for the internal positive controls.
It is well known that when the amount of input DNA in a PCR reaction is reduced below 250 pg, the results can show stochastic effects, such as peak imbalance and allele dropout (19
). All samples in this study that had more than 150 pg added to the PCR reaction gave successful amplification of all 16 STR loci; this included all of the silica method extracts. On the other hand, 10 organic extracts had from 60-150 pg added to the PCR reaction (in a template volume of 10 μL); 4 of these gave successful amplification for all 16 loci, 4 for at least 13 loci (but fewer than 16), and 2 gave seriously deficient partial profiles. Samples 9100990 and 9102658 did not give full profiles with the organic method, but since they were substantially inhibited (with Cts elevated by three and five cycles, respectively, compared to the silica method), it is not known if low DNA, inhibition, or both contributed to the lack of full results.
In conclusion, our experience with large numbers of bone samples showed that it was difficult to predict DNA yield or profiling success from the appearance or characteristics of bones. Consistent with this, the samples that gave lower amounts of DNA or partial profiles showed no characteristics with regard to morphology or environmental context that would permit speculation as to why they may have failed, or why the correlation between yields of the two methods varied among samples. However, given a representative sampling of bones, it is clear that the higher level of DNA recovery with the silica method in some cases is essential to recovering a full DNA profile. The ability to successfully recover nuclear STR profiles from degraded skeletal remains has huge applicability to forensic identification efforts in mass disasters, terrorist attacks, or mass graves. It is important to compare and document the characteristics and efficiency of various DNA extraction methods in order to provide the basis for the selection of the most successful techniques and to establish the capabilities and limitations of methods applied. The silica extraction method developed by the ICMP demonstrated a high success rate on a wide variety of challenging samples. This method performed substantially better in terms of DNA yield, absence of inhibitors, and success in STR profiling than a standard organic extraction approach.