Sylvain Amory

Automatable full demineralization DNA extraction procedure from degraded skeletal remains

During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGENs Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop a more efficient extraction protocol for bone samples that uses significantly less starting material while increasing the success in obtaining DNA results from smaller, more challenging samples. In this study we compared the performance of ICMPs original protocol against an automatable full demineralization approach. In order to provide reliable results and to simulate a wide variety of cases, we analyzed 40 bone samples in a comparative study based on DNA concentrations and quality of resulting STR profiles. The new protocol results in the dissolution of the entire bone powder sample, thus eliminating the possibility that DNA is left behind, locked in remaining solid bone matrix. For the majority of samples tested, the DNA concentrations obtained from half a gram of fully digested bone material were equivalent to or greater than the ones obtained from 2g of partially demineralized bone powder. Furthermore, the full demineralization process significantly increases the proportion of full profiles reflecting the correlation with better DNA quality. This method has been adapted for the QIAcube robotic platform. The performance of this automated full demineralization protocol is similar to the manual version and increases overall lab throughput. It also simplifies the process by eliminating quality control procedures that are advisable in manual procedures, and overall reduces the chance of human error. Finally we described a simple and efficient post-extraction clean-up method that can be applied to DNA extracts obtained from different protocols. This protocol has also been adjusted for the QIAcube platform.

The mtDNA composition of Uzbekistan: a microcosm of Central Asian patterns

In order to better characterize and understand the mtDNA population genetics of Central Asia, the mtDNA control regions of over 1,500 individuals from Uzbekistan have been sequenced. Although all samples were obtained from individuals residing in Uzbekistan, individuals with direct ancestry from neighboring Central Asian countries are included. Individuals of Uzbek ancestry represent five distinct geographic regions of Uzbekistan: Fergana, Karakalpakstan, Khorezm, Qashkadarya, and Tashkent. Individuals with direct ancestry in nearby countries originate from Kazakhstan, Kyrgyzstan, Russia, Afghanistan, Turkmenistan, and Tajikistan. Our data reinforce the evidence of distinct clinal patterns that have been described among Central Asian populations with classical, mtDNA, and Y-chromosomal markers. Our data also reveal hallmarks of recent demographic events. Despite their current close geographic proximity, the populations with ancestry in neighboring countries show little sign of admixture and retain the primary mtDNA patterns of their source populations. The genetic distances and haplogroup distributions among the ethnic populations are more indicative of a broad east–west cline among their source populations than of their relatively small geographic distances from one another in Uzbekistan. Given the significant mtDNA heterogeneity detected, our results emphasize the need for heightened caution in the forensic interpretation of mtDNA data in regions as historically rich and genetically diverse as Central Asia.

DNA Extraction from Aged Skeletal Samples for STR Typing by Capillary Electrophoresis

STR analysis of DNA extracted from skeletal samples can play an important role in the identification of missing persons. Here we present a method for the extraction of DNA from skeletal samples involving complete demineralization and digestion of the sample, followed by purification by silica binding. This method, together with the multiplex STR typing approach also presented, has proven highly successful in the recovery of DNA profiles from degraded, aged skeletal remains from a wide range of environmental contexts. The methodological steps presented include bone decontamination and grinding, DNA extraction, repurification in the case of highly inhibited samples, quantification, STR multiplex amplification, and profile reporting guidelines. However, the conditions applied for amplification and the criteria for allele calling and profile submission must be based on the results of each laboratorys internal validation experiments involving the type of samples relevant to the project at hand. The methods presented here have permitted large-scale DNA-based identification of persons missing from mass disasters and armed conflict.

Prioritized Sampling of Bone and Teeth for DNA Analysis in Commingled Cases

Due to the large number of fragmented and commingled bodies recovered from mass graves related to the war in the former Yugoslavia, DNA typing has become an essential tool for the identification of missing persons from these conflicts. The International Commission on Missing Persons (ICMP) has identified more than 18,500 individuals using DNA analysis of tooth and bone. This wide body of experience permitted an empirical evaluation of the relative preservation of DNA in different skeletal elements. In this chapter, we present the ICMP’s experiences in sampling skeletal remains for DNA analysis, and a resulting detailed protocol for cost-effective DNA-based reassociation in commingled cases.