Nicholas Romanov, also known as Czar Nicholas II, was the last in a line of the Romanov dynasty that ruled Russia for more than 300 years. Nicholas was forced to abdicate his throne at the beginning of the Russian Revolution of 1917. After a brief period of confinement, Nicholas, his entire family, and four servants were executed. The fate of their remains was questioned for nearly 80 years and involved both political and religious debate. A variety of forensic techniques, including mitochondrial DNA analysis , identified the human remains from a pit near Yekaterinburg in the Ural region of Russia as those of the murdered family.
Nicholas Romanov married a German Princess, Alexandra, with whom he had four daughters, Olga, Tatiana, Marie, and Anastasia, and one son, Alexis. His rule of Russia was fraught with domestic and international turmoil. Russia was poorly prepared for World War I and suffered heavy losses. In addition, Alexandra became closely allied with a mystic, Rasputin, who was seen as dangerous by many in the royal court. A series of riots intensified to the level of civil war and Nicholas was forced to abdicate in March of 1917.
After Nicholas was removed from the throne, he and his family were confined. In November of 1917 they were moved from Siberia to the town of Yekaterinburg. On the evening of July 16, 1918, the Romanov family, Alexis' doctor, and three servants were told to dress, as they were to be photographed for a family picture. A Bolshevik execution squad led by Yakov Yurovsky burst into the room, firing shots at the family and their servants. Bullets ricocheted off of jewels that were sewn into the bodices of several of the women. Those who did not die quickly were bayoneted.
The bodies were taken to a spot called Four Brothers, north of Yekaterinburg. They were undressed and the valuables were removed, including about 40 kg of jewels. The bodies were dropped into a deep mine shaft. After word of the killings spread throughout the town, Yurovsky decided to move the bodies to try to better conceal them. Two of the bodies were allegedly set on fire, but this was found to be too time consuming, so the rest were doused with sulfuric acid and buried in a shallow pit about 20 km north of Yekaterinburg.
In 1978 Geli Ryabov, a filmmaker, and Alexander Advonin, a local expert on the executions, decided to try to find the bodies of the Romanov family. They contacted Yurovsky's son, who had a report that his father had written about the murders. It described the location to where the bodies had been moved. Ryabov and Advonin located the burial site on May 30, 1979, and secretly removed two of the skulls. Because of the political situation in the Soviet Union at the time, the two men were unable to provide any further insight into the assassinations, so they reburied the skulls one year later. When the Soviet Union changed its policies to allow for more open exchange of information, Ryabov told the story of the find in 1989. In 1991 Prime Minister Boris Yeltsin called for an official investigation into the origin of the remains.
Approximately 1,000 bones were collected from the burial site. They were reconstructed to form nine bodies, five of which were female and four male. The male skeletons were those of adult men, which suggested that the body of Alexis, who was 13 at the time of his death, was missing. Also missing was the skeleton of one of Nicholas' daughters, though there remained some discrepancy as to which daughter. A Russian team of scientists used a forensic technique called superimposition to identify the skeletons. This technique involves comparing photographic images with skeletal remains to try to link physical features with bone structure. The Russian team concluded that Marie was absent. Using dental comparisons and by study of various bone fragments, a team of scientists from the United States concluded that Anastasia was missing.
In 1992 Pavel Ivanov, a Russian molecular biologist, and Peter Gill of the British Forensic Science Service performed both nuclear and mitochondrial DNA (mtDNA) analyses on the skeletal remains. STR (short tandem repeat) analysis showed that the skeletons belonged to two parents and three female children and four other unrelated people. Prince Philip of England was maternally related to Alexandra and his mtDNA exactly matched the DNA from the skeleton believed to belong to Alexandra.
Results of the mtDNA analysis from the skeleton believed to belong to Nicholas were more difficult to interpret. Nicholas' younger brother Grand Duke Georgij was not alive and the suggestion of exhuming his remains was not an option in 1992. One of Nicholas nephews, Tikhon Kulikovsky, refused to cooperate with the investigation. Eventually, two of Nicholas' distant maternal relatives, Xenia Sfiri and the Duke of Fife, offered to contribute samples of their DNA to the study.
Like nuclear DNA, mitochondrial DNA is made up of a long sequence of four different nucleotides. Mitochondrial DNA analysis compares the sequence of nucleotides in two regions of mtDNA that are highly variable between different people. The mtDNA sequence of Xenia Sfiri and the Duke of Fife matched that of Nicholas except for one single nucleotide. The sequence of mtDNA from bone analyzed from the skeleton believed to belong to Nicholas had a thymine at position 16169, and the mtDNA sequence from Nicholas' relatives had a cytosine at that location.
Additional samples of bone from the skeleton believed to belong to Nicholas were then analyzed to try to reconcile the difference. About 70% of the bone samples contained cytosine at position 16169 and about 30% contained thymine at that location. This variation in mtDNA sequence is known as heteroplasmy and it is exceedingly rare. Some critics claimed that the bone samples must have been contaminated.
In order to convincingly establish whether or not the skeleton actually belonged to Nicholas, the Russian Orthodox Church ordered the body of Nicholas' brother Grand Duke Georgij exhumed in 1994. Analysis of mtDNA from the remains of Georgij resulted in the exact same heteroplasmy as was found in the skeleton believed to belong to Nicholas. Given the rarity of agreement of mtDNA sequence between two people, combined with the unusual occurrence of a heteroplasmy, the probability that the skeletal remains belonged to Nicholas were greater than 100 million to one.
After the source of the remains was established, Nicholas was given a funeral according to the traditions of the Greek Orthodox Church. On July 17, 1998, the remains of Nicholas were laid to rest in the St. Peter and St. Paul Cathedral in St. Petersburg. Two years later, the Church canonized Nicholas, along with his wife Alexandra, stating that their "meekness during imprisonment and poise and acceptance of their martyr's death" deserved great honor.
see also DNA fingerprint; Exhumation; PCR (polymerase chain reaction); Skeletal analysis.