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, 4 (3), e4838

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


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

Michael D Coble et al. PLoS One.


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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Screenshot of the 16169 C/T heteroplasmy present in Tsar Nicholas II using both forward and reverse sequencing primers.
(A) Results from AFDIL's casework section. (B) Results from GMI.
Figure 2
Figure 2. Romanov paternal lineage used for Y-STR testing.
DNA testing for 17 Y-STR markers was conducted on the remains from Tsar Nicholas II and his son, the Tsarevich Alexei (sample 146.1 in the second grave). A distantly related cousin, Prince Andrew Andreevich Romanov of San Francisco, California, was used as a living relative to compare to the skeletal material.
Figure 3
Figure 3. An example of four Y-STR markers from the three Romanov relatives.
Each panel is a screenshot from the blue dye channel of Y-Filer (Applied Biosystems, Foster City, CA). The top panel was developed from the skeletal remains of Alexei, the middle panel was developed from a tooth sample from Tsar Nicholas II, and the bottom panel was developed from Prince Andrew Andreevich Romanov. The loci are (from L to R): DYS456 (16 repeats), DYS389I (13 repeats), DYS390 (24 repeats), and DYS389II (29 repeats).
Figure 4
Figure 4. Screenshots of the 16111 C-T variant and 16357 T-C variants from three samples sequenced from fragments amplified for 440 bp.
Legend for the gel: L = Ladder, RB = Reagent Blank, 7.49 = Sample from Tsarina Alexandra, 146.1 = Sample from Alexei, 5.21 = Sample from Tatiana, NC = Negative Control, PC = Positive Control, L = Ladder.

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