Nine skeletons found in a shallow grave in Ekaterinburg, Russia, in July 1991, were tentatively identified by Russian forensic authorities as the remains of the last Tsar, Tsarina, three of their five children, the Royal Physician and three servants. We have performed DNA based sex testing and short tandem repeat (STR) analysis and confirm that a family group was present in the grave. Analysis of mitochondrial (mt) DNA reveals an exact sequence match between the putative Tsarina and the three children with a living maternal relative. Amplified mtDNA extracted from the remains of the putative Tsar has been cloned to demonstrate heteroplasmy at a single base within the mtDNA control region. One of these sequences matches two living maternal relatives of the Tsar. We conclude that the DNA evidence supports the hypothesis that the remains are those of the Romanov family.

Mitochondrial DNA (mtDNA) from 100 unrelated British White Caucasians was extracted, amplified and directly sequenced. Sequences of approximately 800 nucleotides were obtained from 2 hypervariable segments within the non-coding region of the mitochondrial genome. A total of 91 different sequences were observed with an average nucleotide diversity of 1.1%. The most diverse pair of sequences differed at 3.6% of their nucleotide (nt) sites. Comparison to a consensus reference sequence showed that each region was polymorphic to a similar extent. Different methods of genetic analysis were used to examine the variation in each region, including pairwise comparisons, which demonstrated that although the data did not fit a Poisson distribution, the fit was closer to a Negative Binomial distribution.