Mystery Solved: The Identification of the Two Missing Romanov Children Using DNA Analysis

One of the greatest mysteries for most of the twentieth century was the fate of the Romanov family, the last Russian monarchy. Following the abdication of Tsar Nicholas II, he and his wife, Alexandra, and their five children were eventually exiled to the city of Yekaterinburg. The family, along with four loyal members of their staff, was held captive by members of the Ural Soviet. According to historical reports, in the early morning hours of July 17, 1918 the entire family along with four loyal members of their staff was executed by a firing squad. After a failed attempt to dispose of the remains in an abandoned mine shaft, the bodies were transported to an open field only a few kilometers from the mine shaft. Nine members of the group were buried in one mass grave while two of the children were buried in a separate grave. With the official discovery of the larger mass grave in 1991, and subsequent DNA testing to confirm the identities of the Tsar, the Tsarina, and three of their daughters – doubt persisted that these remains were in fact those of the Romanov family. In the summer of 2007, a group of amateur archeologists discovered a collection of remains from the second grave approximately 70 meters from the larger grave. We report forensic DNA testing on the remains discovered in 2007 using mitochondrial DNA (mtDNA), autosomal STR, and Y- STR testing. Combined with additional DNA testing of material from the 1991 grave, we have virtually irrefutable evidence that the two individuals recovered from the 2007 grave are the two missing children of the Romanov family: the Tsarevich Alexei and one of his sisters

Development of a Multiplex Single Base Extension Assay for Mitochondrial DNA Haplogroup Typing

Cilj Razviti test za brzo pretraživanje, kako bi se skratilo vrijeme testiranja i potrošnja uzorka pri određivanju haploskupina mitohondrijske DNA. Postupci U svrhu vrjednovanja testa ukupno je testirano 147 uzoraka, uključujući 73 uzorka kojima je uspješno određena haploskupina s pomoću tipizacije kontrolnog područja (engl. control region, CR) mitohondrijske DNA (mtDNA), 21 uzorak s nepotpuno tipiziranom haploskupinom na osnovi CR, i 31 uzorak poznatog nasljednog izvora kojima prethodno nije tipizirana haploskupina. Dodatno su s pomoću sustava mnogostruke analize kratkih jezgrenih polimorfnih odsječaka (engl., short nuclear polymorphism, SNP) analizirane dvije jako degradirane ljudske kosti zakopane krajem četrdeseetih godina prošloga stoljeća. Rezultati Kad je mnogostruki sustav SNP primijenjen za tipizaciju 96 uzoraka s prethodno sekvencioniranim CR, opaženo je povećanje definiranja haploskupine ili makrohaploskupine u odnosu na uobičajenu analizu slijeda CR. Prošireni test za pojedinačne baze uspješno je primijenjen u određivanju haploskupina iz uzoraka kostiju starih desetljećima, još iz II. Svjetskoga rata. Zaključak Mnogostruki sustav SNP uspješno je primijenjen za određivanje statusa haploskupina jako raspadnutih ljudskih kostiju, čime je pokazao sposobnost odstranjenja mogućih onečišćenja. Mnogostruki sustav SNP predstavlja jeftin a vrlo učinkovit postupak kojim se mogu tipizirati haploskupine mtDNA A, B, C, D, E, F, G, H, L1/L2, L3, M i N, što znači da se može dobro iskoristiti za brza pretraživanja pri identifikaciji ljudi ili u antropološkim istraživanjima.Aim To provide a screening tool to reduce time and sample consumption when attempting mitochondrial DNA (mtDNA) haplogroup typing. Methods A single base primer extension assay was developed to enable typing, in a single reaction, of twelve mtDNA haplogroup specific polymorphisms. For validation purposes a total of 147 samples were tested including 73 samples successfully haplogroup typed using mtDNA control region (CR) sequence data, 20 samples inconclusively haplogroup typed by CR sequence data, 21 samples previously haplogroup typed using restriction fragment length polymorphism (RFLP) analysis, and 31 samples of known ancestral origin without previous haplogroup typing. Additionally, two highly degraded human bones embalmed and buried in the early 1950s were analyzed using the single nucleotide polymorphisms (SNP) multiplex. Results When the SNP multiplex was used to type the 96 previously CR sequenced specimens, an increase in haplogroup or macrohaplogroup assignment relative to conventional CR sequence analysis was observed. The single base extension assay was also successfully used to assign a haplogroup to decades-old, embalmed skeletal remains dating to World War II. Conclusion The SNP multiplex was successfully used to obtain haplogroup status of highly degraded human bones, and demonstrated the ability to eliminate possible contributors. The SNP multiplex provides a low-cost, high throughput method for typing of mtDNA haplogroups A, B, C, D, E, F, G, H, L1/L2, L3, M, and N that could be useful for screening purposes for human identification efforts and anthropological studies

Armed Forces DNA Identification Laboratory

Abstract In historical cases, missing persons' identification, mass disasters, and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA typing. This is because of the physical and chemical barrier that the protein:mineral matrix of bone poses to environmental deterioration and biological attack. Most bone extraction protocols utilized in the forensic community involve an incubation period of bone powder in extraction buffer for proteinase digestion, followed by the collection of the supernatant, and the disposal of large quantities of undissolved bone powder. Here we present an extremely efficient protocol for recovery of DNA by complete demineralization, resulting in full physical dissolution of the bone sample. This is performed in a manner that retains and concentrates all the reagent volume, for complete DNA recovery. For our study, we selected 14 challenging bone samples. The bones were extracted side-by-side with our new demineralization protocol and the standard extraction protocol in use at AFDIL. A real-time quantification assay based on the amplification of a 143 bp mtDNA fragment showed that this new demineralization protocol significantly enhances the quantity of DNA that can be extracted and amplified from degraded skeletal remains. We have used this technique to successfully recover authentic DNA sequences from extremely challenging samples that failed repeatedly using the standard protocol. Published by Elsevier B.V

CASE REPORT CRIMINALISTICS; ANTHROPOLOGY Integrated DNA and Fingerprint Analyses in the Identification of 60-Year-Old Mummified Human Remains Discovered in an Alaskan Glacier

ABSTRACT: This report describes the identification of a merchant mariner who perished in 1948 when Northwest Airlines Flight 4422, a DC-4 carrying 24 seamen and six crew members crashed into Mount Sanford, Alaska. Fifty-one years later, a human forearm and hand were found close by the wreckage of the plane, prompting identification efforts using DNA and fingerprints. There were significant challenges to both the fingerprint and DNA analyses. The hand was badly desiccated, making fingerprint friction-ridge detail almost invisible and the remains had been embalmed upon discovery, making DNA amplification difficult. We present the results of an interdisciplinary approach that successfully addressed these challenges and ultimately led to the identification of the remains. These efforts relied on efficient fingerprint rejuvenation and imaging techniques that improved print resolution, as well as new DNA extraction techniques optimized for aggressively embalmed remains