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Part three : The tracing of across the millennia by DNA decoding


Every person is a leaf on the paternal and maternal DNA tree. Harvard University geneticist, Professor George Church was a co-founder of the Human Genome Project who once said:” Someday we'll have a complete pedigree of the entire human population. And everybody will be connected to everybody on a huge family tree, like Google maps.”


Once we discovered the secret in our blood expressed as nucleobase letters, we now have a clear and reliable clue to search for the Y chromosome Adam and mitochondrial Eve and thus the origin of our ancestors. 


All these endeavors were originated from our genetic messages. Moreover, our genetic messages come from replications of DNA expressed as nucleobase letters passed down from generation to generation. 

These replicated nucleobase letters retained authenticity, avoided aberrations but on occasion added genetic markers which can help to distinguish different ethnic groups.  Markers on the Y chromosome and mitochondria become the milestones and signposts of tracing our genealogy.


At this juncture, let us tell 3 famous search stories: (a) the “search for Russia’s last princess” (happened in the early 20th century); (b) the 200 years old mysterious search “for Jefferson’s illegitimate child”;  and (c ) the search for China’s Premier Cao Cao’s Y chromosome” (happened 1800 years ago). All these searches   rely on these “milestones and signposts” abovementioned.  And then when we will search for the “Y chromosome Aaron” (happened 3,300 years ago) it will not seem like a fairy tale from “The Arabian Night”.


I. Searching for Russia’s  Last Princess 

1. In the Name of Revolution

Tsar Nicholas II was the last ruler of Russia’s Romanov’s dynasty. In 1917, Nicholas II was forced to abdicate his throne during the turbulent Russian revolution. It was said that to thoroughly crushed the desire of the royalists to restore their former glories, Bolshevik leader Lenin gave the order to execute the tsar and his family.  The executioner, Yekaterinburg, received this order on July 17, 1918 at 1 a.m. The imprisoned tsar, including his family and their four attendants were immediately wakened. The tsar family included Tsar Nicholas II, Empress Alexandra and their four daughters: 23 year old Olga, 21 year old Tatiana, 19 year old Maria and 17 year old Anastasia and 14 year old son Alexei. The four attendants were Dr. Eugene Botkin, male servant Alexei Trupp and female servant Anna Memidova and the cook Ivan Kharitonov. At first, they were told they to move to the basement downstairs for the sake of their safety. However, when they arrived in the basement, a car which normally carried corpses was waiting for them in the courtyard.


 “When they got down to the cellar, they were still not alarmed to find several guards had joined them. Even when they were asked to line up in a group, they were not suspicious. Then the leader of execution squad approached the Tsar and took a piece of paper out of his pocket with one hand while his other rested on a revolver inside his jacket. Hastily he read the notice which condemned them to death. The Tsar was confused. He turned to his family, then to the guards, who drew their weapons. The girls started to scream. The firing began. First to be hit was Tsar; he slumped to the floor. The cellar echoed with the screams as they ricocheted around the room. It was pandemonium, and the room soon filled with smoke, making it even hard for the squad to pick out their target who were rushing to and fro in a blind panic. The order to cease firing was given and the victims were finished off with bayonets and rifle butts.”


These beautiful ladies, handsome young man, innocent attendants instantly became bloody corpse all in the name of revolution.


The above is an excerpt from the book entitled: “The Seven Daughters of Eve”. Seventy years after this mass murder, Brian Skyes, a molecular anthropologist, was asked by the new Russian government to confirm these people’s identities by using mitochondrial DNA from the corpses. He must be told a certain amount of truth. Therefore, the aforementioned events must be reliable.


Figure 3-1: Russian Tsar Nicholas II, Empress, and their five children


The original plan was to bury the 11 executed persons in an old mine. However, the car broke down

halfway on its journey. Hence, with the bodies burnt and their faces disfigured with sulfuric acid, they were hastily buried in a swallow hole. With the passage of time, the whereabouts of the Tsar and his family became a mystery. The Bolshevik Red government merely announced that the Tsar had been executed and failed to mention what had happen to his family or attendants. Therefore, there exists the legend that these people might have escaped the massacre.


Several decades after 1918, there were more than 200 women who claimed to be the last princess Anastasia.  Among these was Anna Anderson who looked very much like the little princess. In 1920, she claimed that she was that princess Anastasia and that she somehow managed to escape on the day of execution.  This had caused exaggerated publicity in the media. Her story even became a 1956 Hollywood film “Anastasia” played by Ingrid Bergman. Nonetheless, there continued to be an inextricable cloud of mystery surrounding the truth as to  what exactly happened to the fate of the Tsar and his five daughters.


On October 1, 2008, the Russian Supreme Court officially ruled that Nicholas II and his family were victims of Soviet repression and should be rehabilitated.



2. Finding the Remains of the Victims


In 1978, two amateur archaeology enthusiasts Charlie Leahbove and Alexander Avtonin  found the burial site based on Nicholai Sokolov’s record. At first, they kept quiet on this finding. Only after the collapse of the Soviet Union in 1989 before they announced the finding to the media.  In 1991, Russia’s General Prosecutor’s office formally excavated the burial ground and discovered 900 pieces of bones. A research team consisting of the British Forensic Science Service, The Russian Academy of Science and the Department of Biological Anthropology at Cambridge University, began the analysis of these remains. Initially, the forensic experts did genetic fingerprinting on the nine corpses. They confirmed two older women who could be the Empress and her attendant and three younger women who could be the Tsar’s daughters.



In the UK, Bryan Sykes, molecular geneticist, an expert in mitochondrial confirmation, and author of” The Seven Daughters of Eve”, was in charge of mitochondrial DNA confirmation from these skeletons. Within a short period of time, he discovered that four out of five of the female’s mitochondrial DNA had similar mutation markers. Dr. Sykes and colleagues published these findings in an article entitled” Identification of the remains of the Romanov family by DNA analysis  in the February 1994 “Nature Genetics”( Reference 1).


Among the skeletons of the four males, one could be the Tsar, the other three could be the doctor, male attendant and the cook. Of course that was only a preliminary assumption. There remains a question, namely if they were part of the group of people executed along with Tsar and his family, then why are we missing two individual. It is really true that there were those who escape the massacre?


In 2007, a group of amateur archaeology enthusiasts found bones 70 meters from the first burial site.  Excavations from the Archeological Institute of the Sverdlovsk region uncovered 44 bones and teeth. These bones could be classified into two: a young man age between 12 to 15 years old and a woman age between 15 to 19 years old. One had a silver-inlaid tooth indicative of nobility. Another finding was that these bones were buried over 60 years ago.  The Russian government invited the U.S. Army Institute of Forensic Medicine, world renowned for their excellent work on archeological DNA confirmation.  Also invited was the Austrian Forensic Institute to work of the mitochondria, autosomes and Y chromosome from the aforementioned two excavations of the 11 persons.  In order to ensure scientific reliability and avoid cross contamination of results, these two laboratories did their research separately. 


In the March 2009 issue of International Journal of Network (PLoS ONE), 13 scientists from aforementioned laboratories from the U.S. Austria, UK and Norway published an article entitled: Mystery Solved: The Identification of the Two Missing Romanov Children Using DNA Analysis”( Reference 2). This article finally found a reliable answer to the 90 year old mystery. The content of what follows come from these two reports.


3. Confirmation from mitochondrial analysis


The Tsar’s mitochondria came from his grandmother, Queen of Denmark Lousie Hesse-Cassel. The empress’s mitochondria came from her grandmother, the British Queen Victoria.  Therefore, their mtDNA’s control regions (high mutational change region) were quite different. These are the maternal ”record of recent historical DNA files”. Therefore, it makes it quite convenient for confirmatory analysis of mtDNA.


The mitochondria of the empress and her five children came from the skeletons. Queen Elizabeth’s husband Prince Philip is the only living person who shares the same matrilineal linkage as the Tsar’s Empress.  On the mtDNA of the empress five children, the same mutation markers occur on the same 3 loci: A263G, C16111T, and T16357C. The rest of the skeletal mtDNA did not have these genetic markers. Therefore, from the mtDNA analysis, the identity of the empress and her children were confirmed. For the reader’s ease of comprehension, the following diagram still uses the “mitochondrial necklace” concept as illustration. This is a good example of determining the maternal relationship by using “mitochondrial necklace” DNA .


The following Figure is based on the combination and simplification of the two reports from 1994 and 2009  to show the maternal hereditary linkage from confirmatory mitochondrial DNA analysis.


Figure 3-2 Empress Alexandra’s Maternal Mitochondrial DNA Genealogy Map


At the very top of the Figure one sees that Princess Alice had two daughters: Empress Alexandra and Princess (later Queen) Victoria. They each passed on their mother’s mitochondrial DNA with crystal clear manifestations of the colored-pearls on the mitochondrial necklace.


4. Confirmation of the Autosomal Chromosomes


The confirmation of autosomes is based on short tandem repeats (STR).  It can be seen that on the five children’s D351358, etc. four DNA locus there were two STRs which were also found in their parents. For example  in daughter Princess Olga’s D351358 STRs  were 17 and 18 with STR 17 from her father and STR 18 from her mother. The other three autosomal loci also matched thus proofing that Princess Olga was the Tsar’s daughter.

Figure 3-3 Simplified results from the analyses of autosomal STR from the remains of the Tsar’s family


Just from the analyses of mitochondria and autosomes, scientists can then reveal the identity of the victims and debunk the 200 individuals who claimed to be Princess Anastasia.

Explanations: 1. Sex chromosome XY indicates male; XX for female; 2. Autosomal loci D3S1358, D16SS39, CSF1PO, D8S1179,  the children must possess the same STR sequences as their parents. For example on the D3S1358 locus, Princes Olga had STR 17 from her father and STR 18 from her mother; Prince Alexie had STR14 from his father and STR 18 from his mother etc.


 5. Confirmation from the Y Chromosome


The aforementioned report also showed a final conclusive evidence as the results of comparing the earlier blood sample from the Tsar when his was young.  On April 29, 1891, during the Tsar’s visit to Japan Otsu city, one of the police men protecting him was assassinated. Fortunately, his life was spared but he suffered two cuts on his head. The shirt he wore on that day was preserved at the Hermitage Museum in St. Petersburg.  From the DNA analysis of the blood stains on his shirt, the Tsar’s autosome and Y chromosome STRs were found to be exactly the same as those found in his skeleton. At the same time, researchers from San Francisco found the grandson of the cousin of the Tsar’s father, Andrew Andreevick, whose Y chromosome was similar to those found on the Tsar’s shirt and skeleton.


At this point, all the confirmatory task has been completed. We could put a period at the end of  the story “search for the last Russian princess”.




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