Different Methods For Measuring mtDna Variation

Over the past two decades, methods for measuring mtDNA variation have progressed in their ability to separate unrelated and closely related maternal lineages. The first studies with mtDNA in the 1980s involved low-resolution restriction fragment length polymorphism (RFLP) analysis using five or six restriction enzymes (see Richards and Macaulay 2001). Higher resolution restriction analysis involved polymerase chain reaction (PCR) amplification of typically nine overlapping fragments followed by digestion with 12 or 14 restriction enzymes. These restriction endonucleases included AluI, AvaII, BamHI, DdeI, HaeII, HaeIII, HhaI, HincII, HnfI, HpaI, MspI, Mbol, RsaI, and "%I (Torroni et a. 1996).

Identifying the remains of the last Russian Czar

Russian Czar (or Tsar) Nicholas II and his family were removed from power and murdered during the Bolshevik Revolution of 1918. They were shot by a firing squad, doused with sulfuric acid to render their bodies unrecognizable, and disposed of in a shallow pit under a road. Their remains were lost to history until July 1991 when nine skeletons were uncovered from a shallow grave near Ekaterinburg, Russia. A number of forensic tests were attempted involving computer aided reconstructions and odontological analysis, but as the facial areas of the skulls were destroyed, classical facial identification techniques were difficult at best and not conclusive.

The Chief Forensic Medical Examiner of the Russian Federation turned to the Forensic Science Service in the United Kingdom to carry out DNA-based analysis of the remains for purposes of identification. Five STR markers (VWA, TH01, F13A1, FES/FPS, and ACTBP2) were used to examine the nine skeletons. Approximately one gram of bone from each of the skeletons yielded about 50 pg of DNA, just enough for PCR amplification of several STR markers. The remains of the Romanov family members consisting of the Tsar, the Tsarina, and three children were distinguishable from those of three servants and the family doctor by their STR genotypes.

While the STR analysis served to establish family relationships between the remains through comparing matching alleles, a link still had to be made with a known descendant of the Romanov family to verify that the remains were indeed those of the Russian royal family. Mitochondrial DNA analysis was used to answer this question.

Mitochondrial DNA was extracted from the femur of each skeleton and sequenced. Blood samples were then obtained from maternally related descendants of the Romanov family and sequenced in the same manner. His Royal Highness Prince Philip, Duke of Edinburgh and husband of the present British Queen Elizabeth, is a grand nephew of unbroken maternal descent from Tsarina Alexandra. His blood sample thus provided the comparison to confirm the sibling status of the children and the linkage of the mother to the Tsarina's family. The sequences of all 740 tested nucleotides from the mtDNA control region matched between HRH Prince Philip and the putative Tsarina and the three children.

The mtDNA sequence from the putative Tsar was compared with two relatives of unbroken maternal descent from Tsar Nicholas II's grandmother, Louise of Hesse-Cassel. The two relatives had the same mtDNA sequence as the putative Tsar with the exception of a single nucleotide at position 16169. At this position, the putative Tsar's sample had a mixture of two nucleotides (T and C), a condition known as heteroplasmy, while the blood samples of relatives had only a T nucleotide.

To further confirm the putative Tsar's remains, the brother of Nicholas II, Grand Duke of Russia Georgij Romanov, was exhumed and tested by the Armed Forces DNA Identification Laboratory (Ivanov et al. 1996). Heteroplasmy was found again at the identical nucleotide site within the mtDNA sequence. Due to the extreme rarity of this heteroplasmy happening by chance between two unrelated individuals, the remains of Tsar Nicholas II and his family were declared authentic and laid to rest in Red Square with a funeral fit for a royal family. However, in spite of the matches made between multiple individuals that were confirmed in a second laboratory, controversy still arises from time-to-time regarding the Romanov remains (see Stone 2004).

Lineage of Romanov Family. The individuals represented by blue are maternal relatives of the Tsarina Alexandra while those shown in red are maternal relatives of Tsar Nicholas II. Living maternal relatives Prince Philip (for Tsarina) and Xenia Cheremeteff-Sfiri (for Tsar) served as family reference samples. The mtDNA mitotype for each reference sample is listed with the nucleotide changes relative to their position in the Anderson sequence. Tsar Nicholas II and his brother Georgij Romanov both exhibited a heteroplasmic T/C at mtDNA position 16169, which differed from the homoplasmic T found in Xenia Cheremeteff-Sfiri (Ivanov et al. 1996). Prince Philip's mitotype matched the remains of the Tsarina and her children while Xenia's mitotype matched the remains of the Tsar at all positions except the heteroplasmic position 16169 (Gill et al. 1994).


Louise of Hesse-Cassel


Tsar Nicholas II

Tsarina Alexandra

Xenia Cheremeteff-Sfiri

Mitotype 16126C 16169T 16294T 16296T 73G 263G 315.1C

Mitotype 16111T 16357C 263G 315.1C

Prince Philip Duke of Edinburgh


Gill, P., Ivanov, P.L., Kimpton, C., Piercy, R., Benson, N., Tully, G., Evett, I.,

Hagelberg, E. and Sullivan, K. (1994) Nature Genetics, 6, 130-135. Ivanov, P.L., Wadhams, M.J., Roby, R.K., Holland, M.M., Weedn, V.W. and

Parsons, T.J. (1996) Nature Genetics, 12, 417-420. Stone, R. (2004) Science, 303, 753.


Genetically different population types or haplotypes have been defined in the literature based on site losses or site gains with the various restriction enzymes. For example, haplogroup A, which is found in Asians and Native Americans, is defined by a site gain at position 663 with Haelll (listed as +663 HaeIII). Haplogroup B was initially defined as a 9 bp deletion in the intergenic region between the COII and tRNALYS genes (see Table 10.2). Individuals belonging to haplogroup A may also be defined by control region polymorphisms 16223T, 16290T, and 16319A while haplogroup B individuals differ from the Anderson reference sequence at 16189C and 16217C.

In the early 1990s, DNA sequence analysis from portions of the control region came into wide acceptance. Most population data outside of the forensic community continues to be collected for only hypervariable segment I (HVS-I) spanning approximately mtDNA nucleotide positions 16024 to 16365. As will be seen below, the forensic DNA typing community has standardized on specific portions of the control region for most of the data that currently exists.

December 2000 marked the beginning of the mtDNA population genomics era with the publication of 53 entire mtGenomes from a diverse set of individuals representing populations from around the world (Ingman et al. 2000). As of early 2004 about a thousand complete mtGenomes exists in public DNA databases (Ruiz-Pesini et al. 2004).

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