Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves

Aim

To quantitatively compare a silica extraction method with a commonly used phenol/chloroform extraction method for DNA analysis of specimens exhumed from mass graves.

Methods

DNA was extracted from twenty randomly chosen femur samples, using the International Commission on Missing Persons (ICMP) silica method, based on Qiagen Blood Maxi Kit, and compared with the DNA extracted by the standard phenol/chloroform-based method. The efficacy of extraction methods was compared by real time polymerase chain reaction (PCR) to measure DNA quantity and the presence of inhibitors and by amplification with the PowerPlex 16 (PP16) multiplex nuclear short tandem repeat (STR) kit.

Results

DNA quantification results showed that the silica-based method extracted on average 1.94 ng of DNA per gram of bone (range 0.25-9.58 ng/g), compared with only 0.68 ng/g by the organic method extracted (range 0.0016-4.4880 ng/g). Inhibition tests showed that there were on average significantly lower levels of PCR inhibitors in DNA isolated by the organic method. When amplified with PP16, all samples extracted by silica-based method produced 16 full loci profiles, while only 75% of the DNA extracts obtained by organic technique amplified 16 loci profiles.

Conclusions

The silica-based extraction method showed better results in nuclear STR typing from degraded bone samples than a commonly used phenol/chloroform method.Analysis by nuclear short tandem repeats (STR) has been proven invaluable for identifications in mass fatality incidents such as plane crashes, terrorist attacks, natural disasters, armed conflict, or any other case where traditional methods of identification are insufficient (1-4). DNA-STR testing often provides the strongest evidence of identity in cases of high degradation of human remains.Bone and teeth are excellent sources of DNA for human identity testing which uses STRs or mitochondrial DNA (mtDNA) (5-7), and in cases of extreme degradation may be the only suitable material available for successful typing. However, relatively specialized techniques are required for the extraction of DNA from bone, particularly when the bones have been exposed to adverse environmental conditions and DNA is degraded and/or present in low amounts. The physical and chemical barriers in bone that protect the DNA from environment and microbial assault also hinder the access of reagents in the extraction process (8,9). Another major difficulty is co-extraction of compounds inhibitory to the polymerase chain reaction (PCR) (10-13), particularly with skeletal samples exposed to soil or other environmental contaminants.It is beyond the scope of this article to review the wide variety of DNA extraction methods that have been reported for bone, but two of the major approaches, with innumerable variations, are 1) organic extraction methods involving phenol/chloroform (7,14,15) and 2) silica-binding methods where guanidinium-based chaotropic salts are used both to disrupt proteins, as well as mediate highly specific binding of DNA to silica particles via ionic salt bridges (12,16,17). Because of the high specificity of DNA binding to silica, inhibition is often less of a problem with this method, and silica binding purifications are sometimes used as secondary clean-up steps after organic extractions to remove inhibitors (15).Nuclear STR profiling has been found highly successful by the International Commission on Missing Persons (ICMP) in their large-scale effort to identify skeletal remains from mass graves in the former Yugoslavia (5,6). In this work, the ICMP has used a silica-binding method based on substantial protocol modifications of a commercially available DNA extraction kit. This DNA extraction method was also used very successfully on more than 1800 bone and tooth samples from the 2004 Asian tsunami that were processed by the ICMP (our unpublished data).In this article, we compared the quantitative performance of the ICMP-developed silica extraction method with a standard organic phenol/chloroform extraction method on the same set of femur samples. Also, we evaluated the levels of PCR inhibitors present in the extract, as assessed by quantitative PCR with internal controls and the amplification obtained with multiplex nuclear STR testing. This comparative study will hopefully assist other groups in adopting simple and effective protocols for DNA isolation from degraded bone in casework relating to mass disasters, terrorist attacks, or mass graves.